Thermostable reverse transcriptase

ABSTRACT

Embodiments of the disclosure concern methods and compositions related to generation and/or use of proofreading reverse transcriptases, including those that are thermophilic or hyperthermophilic. The disclosure encompasses specific recombinant polymerases and their use. In some embodiments, the polymerases are utilized for RNA sequencing in the absence of generation of a cDNA intermediate.

This application is a continuation of U.S. patent application Ser. No.15/410,211, filed Jan. 19, 2017, which claims the benefit of U.S.Provisional Patent Application No. 62/280,451, filed Jan. 19, 2016, theentirety of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant no.FA9550-10-1-0169 awarded by the Air Force Office of Scientific Researchand Grant no. HR0011-12-2-0001 awarded by Defense Advanced ResearchProjects Agency. The government has certain rights in the invention.

TECHNICAL FIELD

Embodiments of the disclosure encompass at least the fields of molecularbiology, cell biology, biochemistry, research, medicine, anddiagnostics.

BACKGROUND

Temin and Baltimore's discovery of reverse transcriptase (RT) alteredthe understanding of molecular biology (Temin and Mizutani, 1970;Baltimore, 1970). It demonstrated that genetic information does not flowunidirectionally, from DNA to RNA to proteins, but could also flow inthe reverse direction from RNA back to DNA. RT enzymes were initiallyfound in retroviruses (e.g., Moloney murine leukemia virus (MMLV)) buthave since been discovered in other RNA elements (e.g., group IIintrons, transposable elements) (Boeke and Stoye, 1997) and areprimarily responsible for converting an RNA genome into DNA forintegration into a host's chromosome. Since its discovery, RT hasrevolutionized the understanding of eukaryotic biology enabling theconversion of mature mRNA into cDNA, without the introns present ingenomic DNA. Since these foundational studies, the RT has become aubiquitous tool in molecular biology driving enabling technologies likenext-generation RNA-Sequencing.

All known RTs are derived from a shared common ancestor (Xiong andEickbush, 1990). These enzymes are characteristically mesophilic andlack a proofreading domain (3′-5′ exonuclease), which is thought to bethe cause of their high error rate in vitro (Roberts et al., 1988). As aresult of this, insertion of the correct nucleotide is driven entirelyby Watson-Crick hydrogen bonding and geometry (Kim et al., 2005). Inaddition, the low polymerization temperature has been a notorious issueinhibiting efficient reverse transcription due to RNAs adopting stablesecondary structures at lower temperatures (Klarmann et al., 1993). Incontrast to RTs, high fidelity DNA polymerases have emerged andinnovated biotechnology—enabling unprecedented fidelity and highthermostability.

Monomeric archaeal Family-B polymerases (polB) have been widely adoptedin modern molecular biology due to their hyperthermostability,processivity, and fidelity. These enzymes have clear advantages over RTsbut they have little to no activity on RNA templates. A comparisonbetween two common archaeal enzymes (KOD and PFU) (Takagi et al., 1997;Lundberg et al., 1991) and MMLV RT reveals the wildtype archaeal polBenzymes failed to polymerize over even five RNA bases (FIG. 1A). The DNAspecificity of these polymerases has likely been driven by evolutionarypressures, as these are presumed to be the genome replicating polymeraseand contain mechanisms actively precluding RNA as a substrate (Greagg etal., 1999).

SUMMARY OF THE INVENTION

Embodiments of the disclosure encompass isolated enzymes that haveproofreading activity and that have reverse transcriptase activity.Embodiments of the disclosure also encompass recombinant enzymes thathave proofreading activity and that have reverse transcriptase activity.In specific embodiments, the enzymes are thermophilic orhyperthermophilic. In at least some aspects, the enzymes are derivativesof a wildtype enzyme, such as a wild-type polymerase. In certain cases,the enzymes are mutated in comparison to a particular polymerase, suchas an Archaeal Family-B polymerase. In particular embodiments, theenzymes of the disclosure have proofreading activity and have reversetranscriptase activity, although the enzymes are mutant versions of anenzyme that lacked reverse transcriptase activity. Embodiments of thedisclosure also concern evolved thermostable polymerase capable ofreverse transcription (cDNA synthesis) and PCR amplification.

In particular embodiments, the enzymes are evolved from anotherpolymerase. The evolved polymerase may be derived from ahypothermophilic archaeal DNA polymerase, distinguished by its abilityfor high fidelity DNA synthesis due to a proofreading (error-correcting)domain. The native archaeal DNA polymerase does not utilize RNA as atemplate, preventing its use as a reverse transcriptase. Directedevolution of the polymerase, during development of embodiments herein,yielded a variant capable of efficient reverse transcriptase activity.This differs from current reverse transcriptases because of itshyperthermostability and its functional proofreading domain, leading tosignificantly increased fidelity of the reverse transcription reaction.Aspects of the disclosure regard enzymes derived from polymerases havingstructure(s) or regions that specifically blocked the use of RNA as asubstrate but that have been manipulated (for example, by design) to beable to utilize RNA as a template.

Thus, embodiments of the disclosure include methods of generatingenzymes that exhibit reverse transcription activity from enzymes that donot exhibit reverse transcription activity. Embodiments also encompassmethods of using enzymes that exhibit reverse transcription activitythat are derived from enzymes that do not exhibit reverse transcriptionactivity.

The disclosed polymerases are the first proofreading reversetranscriptase, offering at least three fold improvement in fidelity overexisting reverse transcriptases. The disclosed polymerases alsoefficiently perform long-range reverse transcription PCR (longer than 5kilobase amplification) as a sole enzyme in the reaction. Specificembodiments of the disclosure provide enzymes that produce cDNA from anRNA template at high temperatures (e.g., >50° C., >55° C., >60° C., >65°C., or higher). Thus, particular enzymes of the disclosure have thefollowing characteristics: high themostability, the ability to reversetranscribe RNA templates, including long RNA templates; andproofreading. In certain embodiments, the enzymes may be utilized inpolymerase chain reaction. In some embodiments, the enzymes may utilizeDNA, RNA, modified DNA, modified RNA, or other nucleotide polymers astemplates. In some embodiments, the enzymes are capable of utilizingtemplates comprising modifications such as the following: 2′-Fluoro,2′-O-methyl, 2′-Amino, 2′-Azido, a-L-threofuranosyl nucleic acid (TNA),1,5-anhydrohexitol nucleic acids (HNAs), cyclohexenyl nucleic acids(CeNAs), 2′-0,4′-C-methylene-b-Dribonucleic acids [locked nucleic acids(LNAs)], arabinonucleic acids (ANAs), or 2′-fluoro-arabinonucleic acids(FANAs). In some embodiments, the enzymes herein produce a DNA polymerfrom DNA monomers (e.g., deoxyadenosine triphosphate, deoxycytidinetriphosphate, deoxyguanosine triphosphate, and deoxythymidinetriphosphate) using a suitable nucleic acid template (e.g., DNA, RNA,modified DNA, modified RNA, other nucleotide polymers, etc.). In someembodiments, the enzymes herein are not capable of producing a non-DNApolymer (e.g., RNA, modified DNA, modified RNA, other nucleotidepolymers, etc.) from a DNA template nor from another nucleic acidtemplate (e.g., RNA, modified DNA, modified RNA, other nucleotidepolymers, etc.). In further aspects, an enzyme of the embodiments isactive on a 2′-O-methyl DNA template.

In particular embodiments, the enzymes comprise one or more additionaldomains, such as one or more polymerization enhancing domains. Theadditional domain may have activity as a DNA clamp, although in cases ofthe disclosure the clamp applies to any template that the enzyme canuse. In certain embodiments the additional domain is able to bindnucleotide polymers. In specific embodiments, the additional domaincomprises all or a portion of one or more of DNA-binding protein 7d(Sso7d), Proliferating cell nuclear antigen (PCNA), helicase, singlestranded binding proteins, bovine serum albumin (BSA), and one or moreaffinity tags.

Embodiments of the disclosure also concern methods of generating theenzymes of the disclosure. In specific embodiments, the methods concerndirected evolution of a new family of proofreading reversetranscriptases. In specific embodiments, reverse transcriptioncompartmentalized self-replication is utilized with primers thatcomprise one or more RNA bases such that when the primer primespolymerization to transcribe the polymerase in question (being testedfor reverse transcriptase activity), the resultant polymerase (eachcompartmentalized in a separate vessel) can only utilize theRNA-comprising strand as a template if it is capable of reversetranscription activity. The pool of mutated polymerases from which totest the members for reverse transcription activity may be generated byany suitable mutation methods.

Methods of using the enzymes of the disclosure for a variety ofapplications are encompassed in the disclosure. Methods related tosequencing of nucleic acids may be performed. For example, methods ofconverting mRNA into cDNA may be performed with enzymes of thedisclosure, as are methods of direct RNA sequencing without a cDNAintermediate. Enzymes of the disclosure allow for methods offacilitating reverse transcription of RNAs comprising stable secondarystructures. In certain aspects, the enzymes are utilizable innext-generation DNA/RNA sequencing technologies.

Compositions and methods of the disclosure provide for more extensiveand accurate copying of any RNA population into cDNA, and hence a moreaccurate record of the molecules in that RNA population; thisfacilitates processes that rely on mRNA molecules, including at leastNextGen Sequencing that relies on mRNA templates (i.e., RNASeq).

Embodiments of the disclosure include enzymes that have reversetranscriptase activity and are derived from recombinant ArchaealFamily-B polymerases. The enzymes may also comprise proofreadingactivity and/or thermophilic or hyperthermophilic activity. As usedherein, a transcriptase activity refers to an enzyme capable ofpolymerizing more than 5, 10, 15, 20, 50, 75, 100, 200 or morenucleotides from a particular template. Thus, in some aspects, an enzymeof the embodiments is able to polymerize more than 5 nucleotides from aRNA or 2′-OMethyl DNA template.

Embodiments of the disclosure concern a recombinant ArchaealFamily-B-derived polymerase that is capable of transcribing a templatethat is RNA, modified DNA, or modified RNA. The modified DNA or modifiedRNA may be modified at the 2′ position of a sugar of a component of thetemplate. In specific cases, the modified DNA or modified RNA comprise amodification selected from the group consisting of 2′-Fluoro,2′-O-methyl, 2′-Amino, 2′-Azido, a-L-threofuranosyl nucleic acid (TNA),1,5-anhydrohexitol nucleic acids (HNAs), cyclohexenyl nucleic acids(CeNAs), 2′-0,4′-C-methylene-b-Dribonucleic acids [locked nucleic acids(LNAs)], arabinonucleic acids (ANAs), and 2′-fluoro-arabinonucleic acids(FANAs). In some cases, the polymerase has proofreading activity and/orthe polymerase has thermophilic or hyperthermophilic activity. Infurther aspects, a polymerase of the embodiments lacks proofreading(3′-5′ exonuclease) activity.

In particular embodiments, a polymerase within the scope herein has oneor more mutations compared to a wild-type or other natural ArchaealFamily-B polymerase. The polymerase may have one or more mutationscompared to wild-type KOD polymerase. The one or more mutations are in aregion of the polymerase that induces stalling at uracil residues; oneor more mutations are in a region that recognizes the 2′ hydroxyl oftemplate RNAs; one or more mutations are in a region that directly actswith a template strand; one or more mutations are in a region forsecondary shell interactions; one or more mutations are in a templaterecognition interface region; one or more mutations are in a region forrecognizing an incoming template; one or more mutations are in an activesite region; and/or one or more mutations are in a post-polymerizationregion, in specific embodiments. In some cases, a mutation is in aregion or position in which the polymerase recognizes the 2′ hydroxyl ofa template RNA. At least one mutation may be an amino acid substitution,in at least some cases.

In certain embodiments, a polymerase has an amino acid sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO:1. In some embodiments, a polymerasecomprises at least one amino acid substitution relative to SEQ ID NO: 1.In some embodiments, the at least one amino acid substitutioncorresponds to an amino acid at positions 384, 389, 664, 493, 97, 521,711, or 735 of SEQ ID NO:1, or any suitable combinations thereof. Incertain aspects, a polymerase of embodiments herein comprises one ormore of the amino acid substitutions provided in Table A. In specificembodiments, the amino acid substitution corresponds to an amino acid atposition 97. In some cases, there is an amino acid substitution thatcorresponds to an amino acid at position 490, 587, 137, 118, 514, 381,38, 466, 734, or a combination thereof. In some embodiments, the aminoacid substitution corresponding to position 384 may be to a histidineresidue or an isoleucine residue. In some embodiments, the amino acidsubstitution corresponding to position 384 may be to a phenylalanineresidue, a leucine residue, an alanine residue, a cysteine residue, aserine residue, a histidine residue, an isoleucine residue, a methionineresidue, an asparagine residue, or a glutamine residue. In someembodiments, the amino acid substitution corresponding to position 389may be an isoleucine residue or a leucine residue. In some embodiments,the amino acid substitution corresponding to position 389 may be to amethionine residue, a phenylalanine residue, a threonine residue, atyrosine residue, a glutamine residue, an asparagine residue, or ahistidine residue. In some embodiments, the amino acid substitutioncorresponding to position 664 may be to a lysine residue or a glutamineresidue. In further aspects, a polymerase of the embodiments does notcomprise a substitution as the position corresponding to position 664.In some embodiments, the amino acid substitution corresponding toposition 493 may be to a leucine residue, a cysteine residue, or aphenylalanine residue. In some embodiments, the amino acid substitutioncorresponding to position 493 may be to an isoleucine residue, a valineresidue, an alanine residue, a histidine residue, a threonine residue,or a serine residue. In some embodiments, the amino acid substitutioncorresponding to position 97 may be to any amino acid residue other thanarginine. In some embodiments, the amino acid substitution correspondingto position 521 may be to a leucine. In some embodiments, the amino acidsubstitution corresponding to position 521 may be to a phenylalanineresidue, a valine residue, a methionine residue, or a threonine residue.In some embodiments, the amino acid substitution corresponding toposition 711 may be to a valine residue, a serine residue, or anarginine residue. In some embodiments, the amino acid substitutioncorresponding to position 711 may be to a leucine residue, a cysteineresidue, a threonine residue, an arginine residue, a histidine residue,a glutamine residue, a lysine residue, or a methionine residue. In someembodiments, the amino acid substitution corresponding to position 735may be to a lysine residue. In some embodiments, the amino acidsubstitution corresponding to position 735 may be to an arginineresidue, a glutamine residue, an arginine residue, a tyrosine residue,or a histidine residue. In some embodiments, the amino acid substitutioncorresponding to position 490 may be to a threonine residue. In someembodiments, the amino acid substitution corresponding to position 490may be to a valine residue, a serine residue, or a cysteine residue. Insome embodiments, the amino acid substitution corresponding to position587 may be to a leucine residue or an isoleucine residue. In someembodiments, the amino acid substitution corresponding to position 587may be to an alanine residue, a threonine residue, or a valine residue.In some embodiments, the amino acid substitution corresponding toposition 137 may be to a leucine residue or an isoleucine residue. Insome embodiments, the amino acid substitution corresponding to position137 may be to an alanine residue, a threonine residue, or a valineresidue. In some embodiments, the amino acid substitution correspondingto position 118 may be to an isoleucine residue. In some embodiments,the amino acid substitution corresponding to position 118 may be to amethionine residue, a valine residue, or a leucine residue. In someembodiments, the amino acid substitution corresponding to position 514may be to an isoleucine residue. In some embodiments, the amino acidsubstitution corresponding to position 514 may be to a valine residue, aleucine residue, or a methionine residue. In some embodiments, the aminoacid substitution corresponding to position 381 may be to a histidineresidue. In some embodiments, the amino acid substitution correspondingto position 381 may be to a serine residue, a glutamine residue, or alysine residue. In some embodiments, the amino acid substitutioncorresponding to position 38 may be to a leucine residue or anisoleucine residue. In some embodiments, the amino acid substitutioncorresponding to position 38 may be to a valine residue, a methionineresidue, or a serine residue. In some embodiments, the amino acidsubstitution corresponding to position 466 may be to an arginineresidue. In some embodiments, the amino acid substitution correspondingto position 466 may be to a glutamate residue, an aspartate residue, ora glutamine residue. In some embodiments, the amino acid substitutioncorresponding to position 734 may be to a lysine residue. In someembodiments, the amino acid substitution corresponding to position 734may be to an arginine residue, a glutamine residue, or an asparagineresidue.

In certain cases, a polymerase has an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO:1 and the polymerase has an amino acidsubstitution at one or more of the following positions corresponding toSEQ ID NO:1: R97; Y384; V389; Y493; F587; E664; G711; and W768. Incertain embodiments, the polymerase has one or more of the followingamino acid substitutions corresponding to SEQ ID NO:1: R97M; Y384H;V389I; Y493L; F587L; E664K; G711V; and W768R, in some aspects.

In specific embodiments, a polymerase has an amino acid sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO:1 and the polymerase has an amino acidsubstitution at one or more of the following positions corresponding toSEQ ID NO:1: F38; R97; K118; R381; Y384; V389; Y493; T514; F587; E664;G711; and W768. In some embodiments, the polymerase has one or more ofthe following amino acid substitutions corresponding to SEQ ID NO:1:F38L; R97M; K118I; R381H; Y384H; V389I; Y493L; T514I; F587L; E664K;G711V; and W768R.

In particular aspects, the polymerase has an amino acid sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO:1 and wherein the polymerase has anamino acid substitution at one or more of the following positionscorresponding to SEQ ID NO:1: F38; R97; K118; M137; R381; Y384; V389;K466; Y493; T514; F587; E664; G711; and W768. The polymerase may haveone or more of the following amino acid substitutions corresponding toSEQ ID NO:1: F38L; R97M; K118I; M137L; R381H; Y384H; V389I; K466R;Y493L; T514I; F587L; E664K; G711V; and W768R.

In certain embodiments, the polymerase has an amino acid sequence thatis at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO:1 and wherein the polymerase has anamino acid substitution at one or more of the following positionscorresponding to SEQ ID NO:1: F38; R97; K118; M137; R381; Y384; V389;K466; Y493; T514; 1521; F587; E664; G711; N735; and W768. The polymerasemay have one or more of the following amino acid substitutionscorresponding to SEQ ID NO:1: F38L; R97M; K118I; M137L; R381H; Y384H;V389I; K466R; Y493L; T514I; I521L; F587L; E664K; G711V; N735K; andW768R.

In further aspects, a polymerase of the embodiments lacks 3′ to 5′exonuclease activity. Methods for inactivating exonuclease activity viaengineered disruption of the exonuclease domain are well known in theart (see, e.g., Nishioka et al., 2001, incorporated herein byreference). For example, in some aspects, a polymerase of theembodiments, has an amino acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence ofSEQ ID NO:1 and has an amino acid substitution corresponding to N210(e.g., N210D), to eliminate 3′ to 5′ exonuclease activity. In furtheraspects, has an amino acid sequence that is at least 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO:1 and has an amino acid substitution corresponding to D141 andE143 (e.g., D141A and E143A), to eliminate 3′ to 5′ exonucleaseactivity. In preferred aspects, a polymerase of the embodiments lackinga 3′ to 5′ exonuclease activity further comprises one or more of theamino acid substitution of Table A. Other amino acid substitutions thatdisrupt the 3′ to 5′ exonuclease activity are within the scope herein.

There is provided herein a recombinant Archaeal Family-B polymerase thattranscribes a template that is RNA and has one or more geneticallyengineered mutations compared to a wild-type Archaeal Family-Bpolymerase, the polymerase having an amino acid sequence at least 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1 andin which one or more amino acid residues at a position selected from thegroup consisting of positions Y493, Y384, V389, 1521, E664 and G711 inthe amino acid sequence shown in SEQ ID NO:1 or at a positioncorresponding to any of these positions, are substituted with anotheramino acid residue. In some cases, the polymerase comprises an aminoacid substitution corresponding to position Y493 to a leucine residue ora cysteine residue. In some cases, the polymerase comprises an aminoacid substitution corresponding to position Y493 to a leucine residue.In some cases, the polymerase comprises an amino acid substitutioncorresponding to position Y384 to a phenylalanine residue, a leucineresidue, an alanine residue, a cysteine residue, a serine residue, ahistidine residue, an isoleucine residue, a methionine residue, anasparagine residue, or a glutamine residue. In some cases, thepolymerase comprises an amino acid substitution corresponding toposition Y384 to a histidine residue or an isoleucine residue. In somecases, the polymerase comprises an amino acid substitution correspondingto position V389 to a methionine residue, a phenylalanine residue, athreonine residue, a tyrosine residue, a glutamine residue, anasparagine residue, or a histidine residue. In some cases, thepolymerase comprises an amino acid substitution corresponding toposition V389 to an isoleucine residue. In some cases, the polymerasecomprises an amino acid substitution corresponding to position 1521 to aleucine. In some cases, the polymerase comprises an amino acidsubstitution corresponding to E664 is to a lysine residue. In somecases, the polymerase comprises an amino acid substitution correspondingto position G711 to a leucine residue, a cysteine residue, a threonineresidue, an arginine residue, a histidine residue, a glutamine residue,a lysine residue, or a methionine residue. In some cases, the polymerasecomprises an amino acid substitution corresponding to position G711 to avaline residue. In some cases, the polymerase comprises an amino acidsubstitution at a position R97 in the amino acid sequence shown in SEQID NO:1 with another amino acid residue. In some cases, the polymerasecomprises one or more amino acid residues at a position selected fromthe group consisting of positions A490, F587, M137, K118, T514, R381,F38, K466, E734 and N735 in the amino acid sequence shown in SEQ ID NO:1or at a position corresponding to any of these positions, which issubstituted with another amino acid residue. In some cases, thepolymerase has proofreading activity. In some case, the polymerase lacksproofreading activity. In some cases, the polymerase has thermophilicactivity. In some cases, the polymerase is capable transcribing at least10 nucleotides from a RNA template. In some cases, the polymerase iscapable of transcribing a template that is 2′-OMethyl DNA. In somecases, the polymerase is capable transcribing at least 5 or at least 10nucleotides from a 2′-OMethyl DNA template.

In some aspects a nucleic acid molecule is provided that encodes apolymerase according to any of the embodiments described herein.Likewise, a method is provided for using a polymerase of the embodimentscomprising the step of contacting the polymerase to a nucleic acidtemplate under suitable conditions to produce a polymerized molecule. Insome cases the nucleic acid template is RNA, DNA or is 2′-OMethyl DNA.

In certain cases, polymerases further comprise an additional domain,such as one that does not itself take part in polymerization but haspolymerization enhancing activity. In a specific embodiment, theadditional domain comprise part or all of DNA-binding protein 7d(Sso7d), Proliferating cell nuclear antigen (PCNA), helicase, singlestranded binding proteins, bovine serum albumin (BSA), one or moreaffinity tags, a label, and a combination thereof.

In one embodiment, provided herein there is a method of using apolymerase according to the embodiments, comprising the step ofsubjecting the polymerase to a nucleic acid template under suitableconditions to produce a polymerized molecule. The template may be RNA orDNA or modified RNA or modified DNA. In specific embodiments, the methodlacks generation of a cDNA molecule. In specific embodiments, the methodprovides sequence information for at least part of the template. Incertain cases, the polymerized molecule is sequenced. The nucleic acidtemplate may be part of a population of nucleic acid molecules, such asa genome or transcriptome, for example. In further aspects, a method ofthe embodiments comprises contacting a polymerase described herein withan RNA template to produce a cDNA. In further aspects, the methodfurther comprises amplifying at least a portion of the cDNA molecules topolymerase chain reaction (PCR). In certain aspects, a polymerase of theembodiments is used both to generate the cDNA and amplify the cDNA. Incertain specific aspects, a method herein is used to produce cDNA fromtwo or more distinct RNA molecules (e.g., from a single cell). Forexample, the method can be used to produce cDNA, and optionallyamplified DNA copies, of antibody VH and VL sequence or T-cell receptorchains (TCR). In certain aspects, the method is used to produce pairedantibody VH and VL coding sequences or paired TCR coding sequences.

In one embodiment, there is a method of selecting an enzyme with reversetranscriptase activity, comprising the steps of: a) providing apopulation of nucleic acids that comprise a region that encodes apolymerase, wherein the polymerase may or may not have reversetranscriptase activity, wherein the region that encodes the polymeraseis flanked by a region in the nucleic acid to which a primer binds,wherein the primer comprises one or more RNA nucleotide bases; b)subdividing the pool of nucleic acids into separate vessels, such thateach vessel comprises a nucleic acid member of the population and thepolymerase encoded by the nucleic acid member; c) subjecting the nucleicacid member and the polymerase to suitable conditions to allowpolymerization from the primer to occur to produce a RNA base-comprisingtemplate; and d) assaying for polymerization of a nucleic acid moleculeusing the RNA base-comprising template as template, wherein when thereis polymerization, the polymerase has reverse transcriptase activity. Insome cases, the method further comprises the step of amplifying the RNAbase-comprising template using the polymerase and/or amplifyingmolecules polymerized from the RNA base-comprising template using thepolymerase. In some embodiments, the method further comprises the stepof producing the population of nucleic acids that comprise a region thatencodes the polymerase. The population may be produced by introducingone or more mutations in nucleic acid that encodes the polymerase. Inspecific embodiments, the one or more mutations are introduced in thenucleic acid randomly. The one or more mutations may be introduced bypolymerase chain reaction. The one or more mutations may be introducedin the nucleic acid in a directed manner. In specific embodiments,nucleic acid in which one or more mutations are introduced correspondsto that which encodes an Archaeal Family-B polymerase that lacks reversetranscriptase activity, such as the Archaeal Family-B polymerase is KODpolymerase. In specific embodiments, the primer comprises more than oneRNA nucleotide base, and the primer may comprise all RNA nucleotidebases. In specific embodiments, the polymerase has reverse transcriptaseactivity and is subject to sequencing.

In another embodiments, a kit is provided that comprises a polymerase ofthe disclosure. In specific cases, the kit comprises one or more of thefollowing: vector(s), nucleotides, buffers, salts, and instructions.

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore well below 0.05%. Most preferred is a composition in whichno amount of the specified component can be detected with standardanalytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows directed evolution of reverse transcriptase functionin Archaeal family-B polymerases. 1A, Primer extension reveals ArchaealpolB are sensitive to RNA in the template strand, stallingpolymerization with several repeat RNAs. 1B, Framework for the directedevolution of hyperthermostable reverse transcriptase using reversetranscription compartmentalized self-replication (RTCSR). Libraries ofpolymerase mutants are created, expressed in E. coli and subjected toemulsion PCR. Primers flanking the polymerase are designed with avariable number of RNA bases separating the plasmid annealing portionfrom the recovery tag, allowing a tunable stringency over the course ofevolution. Recovery PCR specifically amplifies polymerases with reversetranscriptase activity. 1C, Structural heat map of conserved residuesfound by deep sequencing over the RTCSR process. Mutated residues thatare more conserved are colored incrementally darker shades. Amino acidresidues that were mutated in over 50% of the population were labeled.Figure was adapted from KOD structure PDB 4K8Z.

FIGS. 2A-2B shows that engineered reverse transcriptases contain activeproofreading domains. 2A, Single cycle primer extension reaction of KODand CORE3 polymerases and their proofreading deficient counterparts, onboth DNA and RNA substrates. Extension reactions were performed withboth matched 3′ primer:templates (purple) or a 3′ dideoxy mistmatch(orange), which must be excised by proofreading before extension canproceed. The primer is denoted by a gray arrow, extended product ingreen, and exonuclease digested primer in red. 2B, Deep sequencing ofreverse transcription reaction on HSPCB gene using the SSCS technique.The error rate was determined by dividing the sum of base substitutionsand indel formation by the total number of bases sequenced. The errorprofile of reverse transcription is shown as a percentage of allpossible mutations.

FIG. 3 demonstrates single enzyme RTPCR on various genes and RNAsamples. Reverse transcription PCR (RTPCR) was performed using KODpolymerase, CORE3, and the proofreading deficient version of CORE3(exo-). Various genes were amplified, two human genes, PolR2A and p532,and rpoC from E. coli. Using a gene specific forward and reverseprimers, various size amplicons were amplified from these genes,demonstrating efficient single enzyme RTPCR.

FIG. 4 shows that five residues (R97, Y384, V389, E664, and G711) werefully randomized by NNS mutagenesis. Three rounds of RTCSR wereperformed and clones were sequenced. The mutations were counted from thesequencing and labeled on the structure. Residues found in the initialselection are labeled in green. Figure adapted from PDB 4K8Z.

FIGS. 5A-5C. 5A, Mutations in the B11 polymerase (yellow) are mappedonto the KOD polymerase (grey with DNA primer:template duplex in blue).Thirty seven mutations were accumulated, many found in the exonuclease.5B, Examination of the active site of the B11 polymerase shows amutation at glutamate 143 to glycine (SEQ ID NO. 4 and 5). 5C,Functional assays reveal B11 polymerase is capable of single enzymeRTPCR of a 500 base pair region of the HSPCB gene, as well as the B11with grafted wildtype proofreading domain. Proofreading activity wasqualitatively measured in a dideoxy-mismatch PCR, which requires removalof a 3′ deoxy mismatch primer before polymerization occurs.

FIG. 6. The designed polymerases based on the B11 scaffold and deepsequencing information were constructed and tested in an RTPCR assay(HSPCB) and proofreading assay. The mutations introduced into thewildtype KOD polymerase are shown for each of the designed reversetranscriptases.

FIG. 7. To assess the DNA polymerization, a PCR was performed usingunmodified primers on a 2.5 kilobase fragment. Proofreading (3′-5′exonuclease activity) was tested by the addition of 3′ deoxy mismatchprimers into the PCR. Only polymerases capable of removing the mismatchcan extend the primer and perform PCR.

FIG. 8. The SSCS method for reverse transcription is outlined above. Instep 1, total mRNA is isolated. Step 2, barcoded gene specific primersare used to perform first strand synthesis and cDNA is isolated. Step 3,the cDNAs are amplified with primers amplifying the cDNAs whilepreserving the barcodes. Step 4, ILLUMINA® MISEQ® 2×250 sequencer pairedend reads are performed enabling multiple reads of the same initialcDNA. Step 5, identical barcodes are binned and used to create aconsensus sequence. Only barcodes that were read over 3 times were usedin the alignment, reducing sequencer mutations by >99%.

FIG. 9. Steady-state kinetics of polymerase variants. Initial rates ofsingle nucleotide (dCTP) incorporation by exonuclease deficientpolymerases were plotted against the concentration of dCTP using DNA orRNA templates. Kinetic parameters were estimated by fitting the data tothe Michaelis-Menten equation. KOD was able to incorporate dCTP onDNA:RNA duplexes. However, the data could not be fit.

FIGS. 10A-10B. 10A, Relative coverage for various intracellular RNAsfrom gliobastoma cells for each reverse transcriptase. 10B, Clustergramof relative expression for the top 500 most expressed RNAs for MMLV,CORE3, and CORE3 (exo-).

FIG. 11. Primer extension reactions were carried out with a 5′ FAMlabeled oligonucleotide with terminator nucleotides (ddGTP, ddATP,ddTTP, ddCTP) at a 25:1 ratio (ddXTP:dXTP). Reactions were performedwith CORE3 exo- to prevent exonuclease cleavage of terminated extensionproducts. The primer:template RNA complex is depicted with the 3′hydroxyl group on the labeled primer (SEQ ID NOS. 6 and 7). Terminationregion (sequenced bases) is shown in red.

FIG. 12. Schematic demonstrates an example of directed evolution ofreverse transcriptases.

FIG. 13. The table shows the results of paired VH:VL coding sequenceamplification using the CORE3 enzyme (“RTX”) versus a conventionalreverse transcriptase (“Quanta”).

FIG. 14. Individual amino acid substitutions we tested for ability toprovide primer extension activity using an RNA template. A base KODenzyme, lacking exonuclease activity (by introduction of the D141A andE143A substitutions), was used as the negative control and backgroundfor testing the effect of individual substitutions of RNA-templatedprimer extension activity. The CORE3 enzyme (“RTX”) is shown as thepositive control. Results of the study show that each of the testedsubstitutions showed enhanced primer extension (RT) activity on a RNAtemplate as compared to the negative control. The Y493L substitutionshowing the most robust activity for a single substitution.

FIG. 15. Primer extension reactions on DNA and 2′ O-methyl DNA templatesusing KOD, KOD exo-, CORE3 (“RTX”) and CORE3 exo- (“RTX exo-”). KODpolymerases were not capable of primer extension on 2′ O-methyl DNAtemplates. RTX enzymes could polymerize across 2′ O-methyl templates,however full length extension products were only obtained with theproofreading deficient RTX.

DETAILED DESCRIPTION

I. Enzymes of the Disclosure

Despite the critical role that reverse transcriptase plays in molecularbiology, inherent limitations exist in known reverse transcriptases—theyare error prone due to their lack of a proofreading domain. The presentdisclosure concerns proofreading reverse transcriptases, at least someof which that are thermophilic or hyperthermophilic. In particularembodiments, the disclosure concerns the directed evolution of a novelfamily of reverse transcriptases derived from a high fidelityhyperthermophilic Archaeal Family-B polymerase. Over the evolutionaryprocess described herein, the template interface of the polymerase wasdramatically mutated, allowing generation of enzymes that comprisedefficient RNA directed DNA polymerase activity. Embodiments of theengineered polymerase are capable of single enzyme reversetranscription-PCR of long RNAs (e.g., >5 kb) at high temperatures (e.g.,68° C.). In some embodiments, the polymerase retains an activeproofreading domain and achieves the highest in vitro fidelity reported.Kinetic analyses demonstrated roughly equal polymerization efficiency onboth DNA and RNA templates, marking a massive shift in specificitycompared to the parental polymerase. The polymerase was also shown to beeasily incorporated into current RNA-Seq platforms, as well as allowingdirect RNA sequencing without a cDNA intermediate. The unique propertiesof this new family of polymerase enables a deeper and more accurateunderstanding of transcriptomics and drive future biotechnologyinnovations.

In some embodiments, exemplary enzymes of the disclosure have theability to generate DNA from a template that comprises RNA bases, eitherin part or in its entirety. In specific embodiments, the enzymes arerecombinant enzymes. In some embodiments, the enzymes have the abilityto use RNA as a template when their parent enzyme from which they werederived (by mutation) lacked such ability. In specific cases, theenzymes that acquire reverse transcriptase activity are able torecognize alternative bases or sugars in a template strand (compared toan enzyme that can only recognize DNA as a template), such as byallowing recognition of a template having uracil instead of thymine andhaving variability at the 2′ position in the ribose ring.

The enzymes of the present disclosure make it easier to melt RNAstructure and generate cDNA copies, in specific embodiments. Althoughthere are other commercially available reverse transcriptases withmodest thermostability, the enzymes of the present disclosure have muchhigher thermostability (e.g., thermostability at temperatures above 50°C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59°C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68°C., 69° C., 70° C., or more) and have proofreading activity. In specificembodiments, the enzymes of the present disclosure are more processiveand/or more primer-dependent, resulting in less promiscuity ingenerating an accurate cDNA imprint of a mRNA population, for example.Because of their proofreading domain, the enzymes of the presentdisclosure generate fewer mutations than other enzymes and provide amore accurate representation of the RNAs present in a given population(including, for example, a sample from one or more individuals,environments, and so forth).

At least some enzymes of the disclosure encompass proofreading activity,which may be defined herein as the ability of the enzyme to recognize anincorrect base pair, reverse its direction and excise the mismatchedbase, followed by insertion of the correct base. Enzymes of thedisclosure may be referred to as comprising 3′-5′ exonuclease activity.Although testing a particular enzyme for proofreading activity may beachieved in a variety of ways, in specific embodiments the enzyme istested by dideoxy-mismatch PCR that necessitates removal of a 3′ deoxymismatch primer prior to polymerization or primer extension reactionswith 3′ terminal deoxy mismatches.

Although certain enzymes of the disclosure may be characterized asreverse transcriptases, in particular aspects the enzymes can utilizeDNA, RNA, modified DNA, and/or modified RNA as a template. Modified DNAand RNA may be referred to as information nucleotide-comprising polymersthat can be replicated enzymatically that contain altered chemicalmodifications to the backbone, sugar or base. In specific cases, themodified DNA or RNA is modified at the 2′ position of a sugar of acomponent of the template. Particular embodiments encompass recombinantArchaeal Family-B polymerases that transcribe a template that is DNA,RNA, modified DNA, or modified RNA.

The enzymes of the disclosure may be generated using a startingpolymerase that lacks reverse transcriptase activity, and in specificembodiments, that starting polymerase is an Archaeal Family-Bpolymerase, such as KOD polymerase. Any number of mutations may begenerated from the starting polymerase and tested for using methods ofthe disclosure. In specific embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 or more mutations are incorporated into a polymerase thatlacks reverse transcriptase activity such that the entirety of mutations(or a sub-combination thereof) are responsible for imparting reversetranscriptase activity to the polymerase that originally lacked it. Themutations may be of any kind, including amino acid substitution(s),deletion(s), insertion(s), inversion(s), and so forth. In specificembodiments, the mutation is a single amino acid change, and the changemay or may not be conservative. Although in some cases the amino acidsubstitution mutation must be to a certain amino acid, in other casesthe mutation may be to any amino acid. Embodiments within the scopeherein are not limited by the means of generating/designing the variousenzymes. While some enzymes are designed via mutations to a startingpolymerase, embodiments herein are not limited to any particularmechanism of action and an understanding of the mechanism of action isnot necessary to practice such embodiments.

In certain embodiments, an enzyme of the disclosure has a specific aminoacid sequence identity compared to a given enzyme, for example awild-type Archaeal Family-B polymerase, such as KOD polymerase(including, for example, SEQ ID NO:1). In specific embodiments, theenzyme has an amino acid sequence that is at least 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence ofSEQ ID NO:1. An enzyme of the disclosure may be of a certain length,including at least or no more than 600, 625, 650, 675, 700, 725, 750,755, 760, 765, 770, 775, 780, 781, 782, 783, or 784 amino acids inlength, for example. The enzyme may or may not be labeled. The enzymemay be further modified, such as comprising new functional groups suchas phosphate, acetate, amide groups, or methyl groups, for example. Theenzymes may be phosphorylated, glycosylated, lapidated, carbonylated,myristoylated, palmitoylated, isoprenylated, farnesylated, alkylated,hydroxylated, carboxylated, ubiquitinated, deamidated, contain unnaturalamino acids by altered genetic codes, contain unnatural amino acidsincorporated by engineered synthetase/tRNA pairs, and so forth. Theskilled artisan recognizes that post-translational modification of theenzymes may be detected by one or more of a variety of techniques,including at least mass spectrometry, Eastern blotting, Westernblotting, or a combination thereof, for example.

Specific examples of enzymes of the disclosure include at least thefollowing:

B11 reverse transcriptase (an example of a derivative of KOD polymerasethat is a hyperthermophilic reverse transcriptase):

(SEQ ID NO: 2) MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYLYALLKDDSAIEEVKKITAERHSTVVTVKRVEKVQKKFLGRSVEVWKLYFTHPQDVPAIMDKIREHPAVIDIYEYDIPFAIRYLIDKGLVPMEGDEELKLLALDIGTPCHEGEVFAEGPILMISYADEEGTRVITWRNVDLPYVDVLSTEREMIQRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFTLGREGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTVNLPIYTLEAVYEAVEGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEEMPMEAQLSRLIGQSLWDVSRSSTGNLVEWELLRKAYERNELAPNKPDEKELARRHQSREGGYIKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKRMKATIDPIERKLLDYRQRAIKILANSLYGYYGYARARWYCKECAESVIAWGREYITMTIKEIEEKYGFKLIYSDTDGFFATIPGAEAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGLFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHKQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIVDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAYGYRKEDLWYQKTRQVGLSARLKPKGT

CORE3 reverse transcriptase (an example of a derivative of KODpolymerase that is a hyperthermophilic proofreading reversetranscriptase):

(SEQ ID NO: 3) MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYLYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIMDKIREHPAVIDIYEYDIPFAIRYLIDKGLVPMEGDEELKLLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRHQSHEGGYIKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKRMKATIDPIERKLLDYRQRAIKILANSLYGYYGYARARWYCKECAESVIAWGREYLTMTIKEIEEKYGFKVIYSDTDGFFATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGLFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHKQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIVDRAIPFDEFDPTKHKYDAEYYIEKQVLPAVERILRAFGYRKEDLRYQKTRQVGLSARLKPKGT

In particular aspects, the enzymes of the disclosure have one or moremutations in at least one of the following regions of a particularpolymerase (here, as it corresponds to SEQ ID NO:1): residues (1-130 and338-372 is N-terminal domain); (131-338 is exonuclease domain); (448-499is finger domain); (591-774 is thumb domain); (374-447 and 500-590 ispalm domain).

In certain embodiments, the enzymes of the disclosure have mutations atparticular amino acids (the position of which corresponds to SEQ IDNO:1, in certain examples) and, in some cases particular residues arethe substituted amino acid at that position. The table below provides anexample of a list of certain mutations that may be present in thedisclosure, and in specific embodiments a combination of mutations isutilized in the enzyme.

TABLE A Amino acid substitutions for polymerase enzymes of theembodiments KOD Position Mutation for RT activity Possible othermutations Y384 H, I F, L, A C, S, H, I, M, N, Q V389 I, L M, F, T, Y, Q,N, H E664 K, Q Y493 L, C, F I, V, A, H, T, S R97 Any mutation I521 L F,V, M, T G711 V, S, R L, C, T, N, H, Q, K, M N735 K R, Q, N, Y, H A490 TV, S, C F587 L, I A, T, V M137 L, I A, T, V K118 I M, V, L T514 I V, L,M R381 H S, Q, K F38 L, I V, M, S K466 R E, D, Q E734 K R, Q, N

In at least some cases, the enzymes have a mutation at R97 as itcorresponds to SEQ ID NO:1. In some cases, two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, eleven or more, twelve or more, thirteen ormore, fourteen or more, fifteen or more, or sixteen or more mutationsfrom this table are present in an enzyme of the disclosure. In specificembodiments, the following combinations are included alone or with oneor more other mutations listed above or not listed above:

Y384 and V389; Y384 and E664; Y384 and Y493; Y384 and R97; Y384 and1521; Y384 and G711; Y384 and N735; Y384 and A490; V389 and E664; V389and Y493; V389 and R97; V389 and 1521; V389 and G711; V389 and N735;V389 and A490; E664 and Y493; E664 and R97; E664 and 1521; E664 andG711; E664 and N735; E664 and A490; Y493 and R97; Y493 and 1521; Y493and G711; Y493 and N735; Y493 and A490; R97 and 1521; R97 and 1521; R97and G711; R97 and N735; R97 and A490; 1521 and G711; 1521 and N735; 1521and A490; G711 and N735; or G711 and A490. In at least some cases, oneor more other mutations are combined with these specific combinations.

In specific embodiments, the polymerase has an amino acid substitutionat one or more of the following positions corresponding to SEQ ID NO:1:

a) R97; Y384; V389; Y493; F587; E664; G711; and W768;

b) F38; R97; K118; R381; Y384; V389; Y493; T514; F587; E664; G711; andW768;

c) F38; R97; K118; M137; R381; Y384; V389; K466; Y493; T514; F587; E664;G711; and W768; or

d) F38; R97; K118; M137; R381; Y384; V389; K466; Y493; T514; 1521; F587;E664; G711; N735; and W768.

Any of the combinations in a), b), c), or d) may include A490, F587,M137, K118, T514, R381, F38, K466, and/or E734. In particularembodiments, the polymerase has one or more of the following specificamino acid substitutions corresponding to SEQ ID NO:1:

a) R97M; Y384H; V389I; Y493L; F587L; E664K; G711V; and W768R;

b) F38L; R97M; K118I; R381H; Y384H; V389I; Y493L; T514I; F587L; E664K;G711V;

and W768R;

c) F38L; R97M; K118I; M137L; R381H; Y384H; V389I; K466R; Y493L; T514I;F587L; E664K; G711V; and W768R; or

d) F38L; R97M; K118I; M137L; R381H; Y384H; V389I; K466R; Y493L; T514I;I521L; F587L; E664K; G711V; N735K; and W768R.

Any of the combinations in a), b), c), or d) may include A490, F587,M137, K118, T514, R381, F38, K466, and/or E734.

II. Generation of Recombinant Enzymes

Methods in the disclosure provide for the generation of enzymes (e.g.,recombinant enzymes) that comprise reverse transcription activity andproofreading activity and, at least in some cases, are thermophilic orhyperthermophilic. The generation of the enzymes occurs uponmanipulation of a parent polymerase that lacks at least reversetranscription activity. Although a variety of methods may be employed toachieve this end, in particular embodiments the methods utilize highthroughput strategies to obtain mutant versions of a parent polymerase,thereby introducing new characteristic(s) to the resultant enzyme. Inspecific embodiments, directed evolution strategies are employed toproduce development of a recombinant enzyme with reverse transcriptaseactivity from a DNA polymerase that normally lacks reverse transcriptaseactivity. Such differences between the recombinant enzyme and the parentDNA polymerase include development by the recombinant enzyme of theability to use RNA as a template, such as by allowing the enzyme torecognize alternative bases or sugars in a template strand (for example,allowing recognition of a template comprising uracil instead of thymineand allowing variability at the 2′ position in the ribose ring).

In particular embodiments, enzymes of the disclosure are generated frommanipulation of a DNA polymerase that normally lacks reversetranscriptase activity by randomly (or in a directed manner, inalternative embodiments) mutating the polymerase at a region, location,or residue(s) associated with one or more of the following: (1) templateentry to the enzyme; (2) polymerization at the active site; and (3)formation and/or maintaining of the nascent duplex.

Production of the mutant enzymes may occur by any suitable means, andfollowing their generation they may be tested for the ability to reversetranscribe one or more test templates. Examples of randomly (forexample) introducing mutations includes by error-prone PCR, or geneshuffling. Directed mutation may occur by site-directed mutagenesis, forexample.

In specific embodiments, a directed evolution strategy to test newenzymes employs reverse transcription compartmentalized self-replication(RT-CSR). As described herein, methods are employed for utilizing afeedback loop comprising a polymerase in an environment that allowsreplication of only the nucleic acid that encodes it. In the presentcase, primers comprising one or more RNA bases are utilized such thatthe expressed polymerase can only be extracted if it is able torecognize a template that comprises RNA nucleotides (see FIG. 12, forexample). A pool of candidate polymerases that may or may not comprisereverse transcriptase activity may be tested with RT-CSR, with eachcompartment (or vessel) comprising a different candidate polymerase.

The method of testing for candidate polymerases with reversetranscriptase activity may also be a step in methods of generating thecandidate polymerases. In some embodiments, candidate polymerases thatmay or may not have reverse transcriptase activity are produced throughmutation of a known polymerase that lacks reverse transcriptaseactivity. The mutations may be incorporated into the polymerase-encodingnucleic acid molecules by any suitable methods to produce candidatepolymerases with reverse transcriptase activity.

In specific embodiments, a parent polymerase that lacks reversetranscriptase activity and that is used for mutating is an ArchaealFamily-B polymerase, and specific examples include at least DNApolymerases from Thermococcus gorgonarius; Pyrococcus furiosus;Pyrococcus kondakaraensis (also known as Thermococcus kodakarensis);Desulfurococcus strain Tok; Thermococcus sp. 9° N-7; Thermococcuslitoralis; Methanococcus voltae; Pyrobaculum islandicum; Archaeoglobusfulgidus; Cenarchaeaum symbiosum; Sulfolobus acidocaldarius;Sulfurisphaera ohwakuensis; Sulfolobus solfataricus; Pydrodictiumoccultum; and Aeropyrum pernix. Enzymes of the disclosure may have atleast 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical tothe amino acid sequence of a polymerase from one of the above-listedorganisms. Although any DNA polymerase may be used as the parent enzymeto which mutations are imparted to obtain proofreading reversetranscriptases, in some cases the enzyme used for modification is KODpolymerase from Pyrococcus kodakaraensis. The protein sequence for thewild-type enzyme is a follows:

(SEQ ID NO: 1) MILDTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYFYALLKDDSAIEEVKKITAERHGTVVTVKRVEKVQKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVIDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADEEGARVITWKNVDLPYVDVVSTEREMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGINFALGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGQPKEKVYAEEITTAWETGENLERVARYSMEDAKVTYELGKEFLPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRQSYEGGYVKEPERGLWENIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKKKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYITMTIKEIEEKYGEKVIYSDTDGFEATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGDVEKAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLKDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTRQVGLSAWLKPKGT.

III. Methods of Use of the Enzymes

Once an enzyme with desired characteristic(s) has been identified, theenzyme may be utilized in a variety of applications for polymeraseactivity that requires or may require reverse transcriptase activity. Insome cases, the enzymes of the embodiments are used in situations wherestandard reverse transcriptases are employed, including at least nextgeneration sequencing applications (applications with the ability toprocess millions of sequence reads in parallel, such as ILLUMINA®(Solexa) sequencing; Roche 454 sequencing; Ion torrent: Proton/PGMsequencing; Pacbio SMRT sequencing, and SOLiD sequencing). The enzyme(s)may be employed particularly when high fidelity is necessary or could benecessary. In this case, polymerase enzymes with proofreadingexonuclease activity would be preferred. In certain embodiments, theenzymes are employed at least for molecular biology applications such asdiagnostics (such as analyzing nucleic acids from a biological sample orderived from nucleic acids from a biological sample); cDNA librarycloning, and next-generation RNA sequencing.

In certain embodiments, one can utilize one or more enzymes of thedisclosure for direct RNA sequencing in the absence of first generatinga cDNA intermediate. Such methods for the disclosure for applying theenzyme(s) (and others) allow beneficial avoidance of bias in generationof cDNA populations and subsequent amplification. In certainembodiments, enzymes of the disclosure have the ability to performdirectional RNA sequencing, which preserves information about strandorientation, such as by the incorporation of dUTP into the first orsecond strand synthesis. Furthermore, in some aspects, enzymes of theembodiments can be used for both reverse transcription and subsequentamplification cDNA by polymerase chain reaction. Thus, in some aspects,reverse transcription-PCR can be performed in a single reaction usingthe same polymerase enzyme.

Further methods that can preferably employ an enzyme of the embodimentsinclude, without limitation:

-   -   Method that require thermal denaturation of components, such as        PCR inhibitors (e.g., proteins or heat sensitive molecules),        prior to reverse transcription.    -   Improved reverse transcriptase and/or polymerization of nucleic        acid species in compartments, such as water-in-oil emulsions.        Such methods could be used to amplify, including pairing two or        more RNA sequences with overlap extension RT-PCR, sequences from        samples including individual cells or tissue samples. For        example, these amplifications may include techniques such as        digital droplet PCR.    -   The 3′-5′ exonuclease activity of polymerases of the embodiments        (e.g., CORE3) can be used to detect single nucleotide        polymorphisms (SNPs) present in RNA or DNA sequences using a        primer mismatch extension assay. These amplification products        can be read out using sequencing and/or direct visualization.    -   Polymerase blends using polymerases of the embodiments (e.g.,        CORE3) with other known RTs (MMLV, AMV, Tth, and other        engineered variants such as Taq polymerase) may provide further        increased performance for the detection of difficult to        synthesize nucleic acid sequences.    -   Polymerases of the embodiments have been demonstrated to work        with multiple template compositions (DNA, RNA, and 2′C-Omethyl)        and should reverse transcribe additional unnatural nucleic acid        compositions, which could be applied to additional therapeutic,        diagnostic, or sequencing applications.    -   Polymerases of the embodiments may be utilized in amplification        schemes where the polymerase serves as a reverse transcriptase        and other polymerases including DNA/RNA polymerases aid in        amplification, such as: RT-Lamp, 3SR (NASBA), transcription        mediated amplification (TMA), RCA, RPA, HDA, Strand displacement        amplification.    -   Molecular cloning methods can also utilize polymerases of the        embodiments such as SLIC, or Gibson assembly such that RNAs can        directly be used or RNA containing primers.    -   Polymerases can be used for high fidelity cDNA library        generation    -   Immuno-PCR amplification techniques can employ polymerases of        the embodiments to detect small molecule or protein metabolites.    -   Polymerases of the embodiments can likewise be used for the in        vitro or in vivo selection of RNA aptamer sequences including        RNA-modified aptamers.    -   In vivo expression of polymerases of the embodiments can be used        to convert RNA in cells into DNA. This could be used for, for        instance for, programmed recombination (e.g., retrons,        retroelements) or storage of nucleic acid information.    -   Polymerases can also be used in selection techniques for        directed evolution including compartmentalized partnered        replication or cooperative-C S R directed evolution techniques.

IV. Kits of the Disclosure

All or some of the essential materials and reagents required forproducing, testing, and/or using enzymes of the disclosure may beprovided in a kit. The kit may comprise one or more of RNAbase-comprising primers, vectors, polymerase-encoding nucleic acids,buffers, ribonucleotides, deoxyribonucleotides, salts, and so forthcorresponding to at least some embodiments of the enzyme production,characterization, and/or use. Embodiments of kits may comprise reagentsfor the detection and/or use of a control nucleic acid or enzyme, forexample. Kits may provide instructions, controls, reagents, containers,and/or other materials for performing various assays or other methods(e.g., those described herein) using the enzymes of the disclosure.

The kits generally may comprise, in suitable means, distinct containersfor each individual reagent, primer, and/or enzyme. In specificembodiments, the kit further comprises instructions for producing,testing, and/or using enzymes of the disclosure.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Strategies have been developed to allow directed evolution ofpolymerases in a high throughput manner, which have successfully led topolymerases with altered specificity and attributes (Pinheiro et al.,2012; Ghadessy et al., 2001). In the present disclosure, there wasadaptation of the directed evolution framework to allow evolution ofalternative bases or sugars in the template strand, a strategy calledreverse transcription compartmentalized self-replication (RT-CSR) (FIG.1B). Briefly, libraries of polymerase mutants are expressed in E. coliand subsequently subjected to emulsion PCR, physically separating cellsinto individual compartments and enabling the amplification of encodedpolymerase inside the bacterium. The primers flanking the polymerase inthe emulsion PCR are designed with RNA bases separating a capturesequence and a plasmid binding sequence. Upon second strand synthesis,the polymerases are challenged with RNA template derived from theinitial elongated primer. The number of intervening RNA bases wasincreased to increase the stringency, allowing incrementally morechallenging templates as the polymerases evolved (Table 1).

TABLE 1 RTCSR selection parameters used during the evolution of polBreverse transcriptases. The number of RNA bases in the RTCSR are denotedin the forward and reverse primers. Total Plasmid Induction aTc Round #Mutagenesis RTCSR Primers RNA Backbone [ng/ml] Initial Library 0 errorprone colE1 (-rop) 1 error prone RTCSR.F/RTCSR.R 0 colE1 (-rop) 200 2error prone RTCSR.F/RTCSR.R 0 colE1 (-rop) 200 3 N/A RTCSR.F/RTCSR.R 0colE1 (-rop) 200 4 N/A RTCSR.RNA5.F/RTCSR.RNA5.R 10 colE1 (-rop) 200 5error prone RTCSR.RNA5.F/RTCSR.RNA5.R 10 colE1 (-rop) 200 6 N/ARTCSR.RNA5.F/RTCSR.RNA5.R 10 colE1 (-rop) 200 7 error proneRTCSR.RNA10.F/RTCSR.RNA5.R 15 colE1 (-rop) 200 8 N/ARTCSR.RNA10.F/RTCSR.RNA5.R 15 colE1 (-rop) 200 KOD97 Library 0 errorprone colE1 (-rop) (R97 NNS) 1 N/A RTCSR.RNA5.F/RTCSR.RNA5.R 10 colE1(-rop) 200 2 error prone RTCSR.RNA5.F/RTCSR.RNA5.R 10 colE1 (-rop) 200 3N/A RTCSR.RNA10.F/RTCSR.RNA5.R 15 colE1 (-rop) 200 4 error proneRTCSR.RNA10.F/RTCSR.RNA5.R 15 colE1 (-rop) 200 5 N/ARTCSR.RNA10.F2/RTCSR.RNA10.R2 20 colE1 (-rop) 200 6 error proneRTCSR.RNA10.F2/RTCSR.RNA10.R2 20 colE1 (-rop) 200 7 N/ARTCSR.RNA15.F2/RTCSR.RNA10.R2 25 colE1 (-rop) 200 8 error proneRTCSR.RNA15.F2/RTCSR.RNA10.R2 25 colE1 (-rop) 200 9 N/ARTCSR.RNA52.F2/RTCSR.RNA10.R2 62 colE1 (-rop) 200 10 N/ARTCSR.RNA52.F2/RTCSR.RNA10.R2 62 colE1 (-rop) 200 11 N/ARTCSR.RNA52.F2/RTCSR.RNA48.R3 100 colE1 (-rop) 200 12 N/ARTCSR.RNA52.F2/RTCSR.RNA48.R3 100 colE1 (-rop) 200 13 N/ARTCSR.RNA52.F2/RTCSR.RNA48.R3 100 p15A 20 14 N/ARTCSR.RNA52.F2/RTCSR.RNA48.R3 100 p15A 20 15 N/ARTCSR.RNA52.F2/RTCSR.RNA48.R3 100 p15A 20 16 N/ARTCSR.RNA52.F2/RTCSR.RNA48.R3 100 p15A 20 17 N/ARTCSR.RNA88.F2/RTCSR.RNA88.R3 176 colE1 (-rop) 200 18 N/ARTCSR.RNA88.F2/RTCSR.RNA88.R3 176 colE1 (-rop) 200

Evolution was initiated by evenly distributing mutations across the KODpolymerase by error prone PCR, as a targeted approach was deemedimpractical due to extensive interactions between the polymerase and thetemplate strand. Initially, a modest selection pressure was used,containing 10 total RNA bases (5 per each priming oligonucleotide), asthis condition exceeded the number of template RNA bases wildtype KODcould polymerize. Upon sequencing the pool after eight rounds, mutationsin position R97 were observed in over 90% of the variants. This generalregion of the polymerase is known to induce stalling at uracil residues(Killelea et al., 2010; Firbank et al., 2008), but unlike the morecommon mutation to inactivate this function (V93Q) (Fogg et al., 2002),analysis made during developments of embodiments herein reveals that R97is positioned to recognize the 2′ hydroxyl of template RNAs. A fullrandomization of this position was then made before continuing theselection.

Another eighteen rounds of selection were performed on the polymerasepool, introducing diversity as needed. The selection pressure wasgradually increased until primers in the selection were completelycomposed of RNA, requiring reverse transcription to occur everythermal-cycle in order to maintain exponential amplification in theemulsion PCR. Deep sequencing of the polymerase pool identified crucialmutations for the adaptation of reverse transcriptase ability (FIG. 1Cand Table 2).

TABLE 2 Deep sequencing of RTCSR libraries. Amino acid residues withmutations occurring in 10% of the population are shown in order offrequency. Some positions contained several amino acid possibilities.Synonymous mutations are not shown. Amino acid Mutation Amino acidVariant position Frequency change Frequency Initial Selection 97 94.2% R→ H 58.70% R → S 22.30% R → C 13.20% 587 27.9% F → L 15.10% F → L 12.80%119 R → H 10.90% Round 10 97 97.9% R → F 17.70% R → A 11.80% Other68.40% 384 Y → H 81.20% 210 N → D 63.70% 389 V → I 50.20% 587 37.3% F →I 14.00% F → L 23.30% 711 G → S 29.30% 664 E → K 29.20% 168 A → T 25.70%521 I → L 24.20% 454 G → D 22.20% 490 A → T 17.40% 634 G → D 16.00% 528I → L 14.50% 734 E → K 14.10% 493 Y → C 13.90% 311 Y → C 12.10% 292 A →T 11.80% 137 M → I 11.30% 677 G → S 10.90% 440 R → H 10.80% 144 T → A10.80% 171 I → V 10.60% 748 F → Y 10.00% Amino Acid Mutation Amino AcidVariant Position Frequency Change Frequency Initial Selection 97 94.2% R-> H 58.70% R -> S 22.30% R -> C 13.20% 587 27.9% F -> L 15.10% F -> L12.80% 119 R -> H 10.90% Round 10 97 97.9% R -> F 17.70% R -> A 11.80%Other 68.40% 384 Y -> H 81.20% 210 N -> D 63.70% 389 V -> I 50.20% 58737.3% F -> I 14.00% F -> L 23.30% 711 G -> S 29.30% 664 E -> K 29.20%168 A -> T 25.70% 521 I -> L 24.20% 454 G -> D 22.20% 490 A -> T 17.40%634 G -> D 16.00% 528 I -> L 14.50% 734 E -> K 14.10% 493 Y -> C 13.90%311 Y -> C 12.10% 292 A -> T 11.80% 137 M -> I 11.30% 677 G -> S 10.90%440 R -> H 10.80% 144 T -> A 10.80% 171 I -> V 10.60% 748 F -> Y 10.00%Round 18 384 Y -> H 96.00% 97 93.3% R -> A 20.80% R -> F 18.00% Other54.50% 389 V -> I 91.90% 210 N -> D 84.90% 493 83.3% Y -> C 59.00% Y ->L 13.20% Y -> F 11.10% 664 82.7% E -> K 60.40% E -> Q 22.30% 711 75.0% G-> S 46.80% G -> V 28.20% 521 I -> L 59.40% 490 A -> T 58.50% 587 55.1%F -> L 36.80% F -> I 18.30% 168 A -> T 36.70% 734 E -> K 34.50% 13733.9% M -> I 20.30% M -> L 13.60% 748 F -> Y 22.40% 735 N -> K 18.80%593 K -> N 16.90% 590 T -> A 15.80% 605 T -> I 13.20% 143 E -> G 13.00%501 R -> H 12.90% 144 T -> A 12.50% 150 E -> D 12.20% 145 L -> P 11.50%741 V -> A 11.30% 692 K -> R 11.20% 454 G -> D 11.10%

The majority of conserved mutations directly interact with the templatestrand, are secondary shell interactions, or are known to inactivate theproofreading activity (N210D). These mutations span the length of thetemplate recognition interface, being situated in the regionsrecognizing the incoming template (R97), the active site (Y384), or postpolymerization—the nascent RNA/DNA duplex (V389, 1521, E664,G711)(Bergen et al., 2013).

Based on the single directed evolution experiment, it was unclearwhether the conserved mutations served to abolish strict DNA specificityor promoted RNA specificity. In attempt to answer this question, theRTCSR process was replayed but this time fully randomizing suspected keyresidues in the parental polymerase. This revealed a potentiallytwo-pronged evolutionary process, the loss of function at certainpositions (e.g., R97) based on many viable solutions and mutationspotentially promoting RNA utilization (e.g. Y384H, E664K) due toevolutionary amino acid preferences (FIG. 4). In the contextual view ofthe wildtype polymerase, the observation that (1) many mutations wererequired for efficient reverse transcription ability and (2) themutations were spread across the entire template interface, suggeststhat the wild-type polymerase utilizes a series of checkpoints todiscriminate DNA from RNA: as the template enters the enzyme, aspolymerization occurs at the active site, and at the nascent duplex.

Screening the pool for active polymerases yielded a variant, B11, with37 mutations. The polymerase was found to be capable of reversetranscription of at least 500 base pairs. Sequencing revealed that thepolymerase had inactivated the proofreading domain, which was confirmedin functional assays (FIG. 5). It was considered that transplantation ofthe wildtype proofreading domain might restore this activity. The hybridrecovered activity, but to barely detectable levels. Despite theproofreading domain regaining activity, the RT activity in the B11polymerase was still robust, indicating that RT activity is compatiblewith 3′-5′ exonuclease activity. Encouraged by these results, severaldesigned polymerases were constructed to minimize what were likelyextraneous mutations likely introduced in the RTCSR process, in certainaspects.

Polymerases were designed based on the B11 scaffold, as well assequencing data of the pool. A series of polymerases were built aroundwhat were likely a core set of mutations, as identified by highlyconserved mutations and residues in proximity to the template. Testingrevealed that each of the polymerases were active but reversetranscriptase activity was enhanced upon additional mutations (FIG. 6).Proofreading activity was demonstrated in each of the core polymerasedesigns—indicating that constructing more wildtype-like polymerases didenhanced proofreading. Based on activity of these polymerases, the CORE3polymerase was chosen to characterize further, as it has the mostsubstantial reverse transcriptase activity while still maintaining itsproofreading capability.

Based on the screening metric, the CORE3 polymerase had proofreadingactivity on DNA templates (FIG. 7) but it was unclear whether theproofreading mechanism could occur during reverse transcription, asRNA:DNA duplexes adopt alternative conformations not found in DNA-onlyduplexes (Wang et al., 1982). To address this, oligonucleotides weresynthesized so that the 3′ end would either form a canonical 3′ hydroxylmatched base pair or a 3′ deoxy mismatched pair—analogous a newlymis-incorporated nucleotide, which can stimulate proofreading activity.When primer extensions were performed using a DNA template, bothparental KOD and CORE3 both were capable of extending the mismatchedprimer in a single extension, while their exonuclease deficientcounterparts were not. Repeating this assay using an RNA template, theCORE3 polymerase did not lose proofreading activity and it wascomparable to the proofreading activity while polymerizing on a DNAtemplate (FIG. 2A).

Given that CORE3 maintains proofreading during reverse transcription,the impact that proofreading could have on reverse transcriptionfidelity was considered. A strategy was devised based on recent advancesthat have significantly reduced ILLUMINA® sequencing errors byimplementing barcodes into adaptors sequences (Schmitt et al., 2012),allowing for precise detection of errors unambiguously, unliketraditional genetic based assays. Unique barcodes were designed intoreverse transcription primers of human HSPCB and PolR2A genes. After RT,a subsequent PCR of the cDNAs copies these barcodes, allowing multiplereads during deep sequencing (FIG. 8). Binning identical barcodes (N≥3)and creating consensus sequences reduces background errors by severalorders of magnitude. Sequencing of the genes revealed the mutationalspectra of MMLV, CORE3 and the proofreading deficient CORE3 (exo-) (FIG.2B and Table 3).

TABLE 3 a HSPCB reverse transcription CORE3 Polymerase CORE3 MMLV exo−B11 Total Matches 1.44E+07 1.20E+06 1.10E+06 1.33E+07 Total Mismatch 520124 102 3136 Total Indel 15 7 11 416 Error Rate 3.71E−05 1.10E−041.03E−04 2.66E−04 Base:Mutation Mutation Frequency T to A 1.92% 2.42%20.59% 23.82% G to A 27.69% 29.03% 13.73% 6.12% T to C 5.58% 22.58%1.96% 2.68% G to C 0.38% 0.00% 0.00% 0.00% T to G 3.27% 0.00% 6.86%8.16% C to G 0.38% 0.81% 0.98% 2.36% C to A 3.46% 3.23% 10.78% 6.92% Ato T 1.54% 9.68% 0.98% 1.66% G to T 31.73% 1.61% 0.98% 4.11% C to T11.35% 13.71% 11.76% 10.01% A to C 0.19% 8.06% 0.98% 0.70% A to G 12.50%8.87% 30.39% 33.45% PolR2A reverse transcription Polymerase CORE3 MMLVCORE3 exo− Total Matches 1.66E+07 1.12E+06 1.26E+07 Total Mismatch 537536 4175 Total Indel 54 7 965 Error Rate 3.56E−05 4.86E−04 4.08E−04Base:Mutation Mutation Frequency T to A 2.61% 0.56% 35.52% G to A 14.34%1.68% 2.18% T to C 13.04% 88.25% 2.68% G to C 0.74% 0.37% 0.05% T to G1.49% 0.00% 1.51% C to G 0.37% 0.19% 2.35% C to A 6.89% 0.00% 5.27% A toT 1.12% 0.19% 2.75% G to T 34.08% 2.05% 3.83% C to T 12.66% 2.80% 8.02%A to C 0.56% 0.75% 1.20% A to G 12.10% 3.17% 34.63% b HSPCB (DNATemplate) CORE3 Polymerase CORE3 MMLV exo− B11 KOD Total Matches1.84E+07 2.23E+06 4.65E+06 2.33E+07 1.49E+07 Total Mismatch 1521 297 7955697 627 Total Indel 305 17 92 852 5 Error Rate 9.93E−05 1.41E−041.91E−04 2.80E−04 4.23E−05 Base:Mutation Mutation Frequency T to A 4.67%5.39% 15.47% 19.89% 2.71% G to A 13.41% 14.14% 14.97% 9.60% 26.16% T toC 3.35% 7.74% 3.40% 3.48% 5.74% G to C 0.13% 0.34% 1.13% 2.42% 0.00% Tto G 0.66% 0.34% 2.14% 3.39% 0.00% C to G 5.85% 1.35% 11.45% 11.01%0.32% C to A 14.73% 10.10% 16.48% 10.88% 34.13% A to T 1.58% 12.46%2.52% 6.90% 0.48% G to T 6.38% 7.41% 2.64% 2.98% 6.54% C to T 12.29%8.08% 9.06% 10.67% 8.29% A to C 0.72% 12.79% 0.75% 1.79% 0.80% A to G36.23% 19.87% 20.00% 16.99% 14.83% a, Fidelity profile for reversetranscription on two human genes, HSPCB and PolR2A using the SSCStechnique. The error rate is calculated by dividing total mutations(mismatch + indel) over the total number of bases sequenced. Thefrequency of each possible mutation is listed as a percentage of totalmutations. b, Fidelity profile for DNA template (cloned plasmid DNA)polymerization using cloned HSPCB.

The MMLV control enzyme had an error rate of 1.1×10⁻⁴ while the CORE3enzyme had an error of 3.71×10⁻⁵ (˜3-fold improvement). Inactivating theproofreading of CORE3 lowers the observed fidelity nearly 3 fold—furthersupporting that CORE3 contains active proofreading while reversetranscribing. In specific embodiments, the true error rate for CORE3 islower given that the SSCS technique has a lower limit of detectionnearly identical to the error rate observed for CORE3—due to artifacts,which was confirmed by experiments measuring wild-type KOD on DNA (Table3)(Schmitt et al., 2012). In addition, transcriptionally derived errorsare unaccounted for and other experiments have demonstrated that anactive proofreading domain can increase fidelity ˜30-fold (Nishioka etal., 2001).

Having seen robust reverse transcription with shorter templates, theCORE3 polymerase was tested in a single enzyme RT-PCR (in which theCORE3 polymerase performs both the first-strand reverse transcription aswell as the PCR amplification) for much longer templates. Severalindependent RNA sources and gene loci were chosen to mitigate thepossibility of contaminating DNA. Eukaryotic mRNAs were emphasized inthe testing, as DNA contamination would create a unique size profile,due retention of introns. Across three unique RNA samples and genes, theCORE3 polymerase was highly capable of single enzyme RTPCR, successfullygenerating amplicons larger than 5 kilobases (FIG. 3). The experimentsindicate that inactivating the proofreading of CORE3 (N210D mutation),which generally increases product yield, was not necessary to achievethese large amplicons.

Having shown that the CORE3 polymerase is capable of reversetranscribing long RNAs with proofreading ability, it was considered howthe evolutionary process enabled such a radical shift in function.Steady state analysis of incorporation of radio-labeled dCTP of theancestral and evolved polymerases reveals a dramatically decreased K_(m)on RNA, from nondetectable substrate binding (in the parental KODenzyme) to affinities comparable to wild-type KOD binding on a DNAtemplate (Table 4 and FIG. 9).

TABLE 4 Steady state kinetics for polymerase variants on DNA and RNAtemplates. DNA RNA Enzyme kcat Km kcat/Km kcat Km kcat/Km KOD 160.4 39.74.0 n.d. n.d. n.d. B11 48.2 7.9 6.1 54.5 23.1 2.4 CORE3 49.1 15.7 3.152.3 51.5 1.0 CORE3 56.8 12.1 4.7 55.2 31.1 1.8 exo- (n.d. was notdetermined due to inactivity)

The K_(m) of the evolved polymerases appeared to be lowered for DNAtemplates as well, which is a general phenomenon while evolving DNApolymerases using CSR (data not shown), presumably to increase productyield in the emulsion PCR reaction. While the exact role of eachmutation in CORE3 is not known, the increased affinity is in part due tothe E664K mutation which was observed in high frequency throughout theevolution experiment, and has been demonstrated to greatly increasebinding DNA/RNA heteroduplexes (Cozens et al., 2012).

The advantages of a high fidelity RT has great potential to increaseunderstanding of transcriptomics, reducing biases and errors introducedin the reverse transcription step of nextgen RNA-Seq. To demonstrate theimmediate utility, the CORE3 polymerase was implemented into a commonlyused work flow for directional RNA sequencing (NEBNEXT® library prep).The workflow was unaltered except the buffer and polymerase were changedto CORE3 in the reverse transcription step. Analysis revealed nearlyidentical coverage and expression profiles (FIG. 10), indicating thatthe proofreading activity of CORE3 does not introduce systematic biasesof mRNA expression levels.

Using the CORE3 polymerase, it was considered that it might be possibleto bypass the need to create cDNA libraries all together, in specificembodiments. The process of cDNA synthesis and PCR amplification haslong been known to introduce many biases through amplification (Hansenet al., 2010; Aird et al., 2011). CORE3 was utilized to directlysequence RNA using traditional Sanger sequencing approach. Singledideoxy-terminator nucleotides were mixed with normal dNTPs, such thattermination would occur partially at each corresponding base, whichcould then be run on a sequencing gel or capillary. As a proof ofconcept, 20 nucleotides of a GATC₅ RNA repeat were sequenced (FIG. 11).Termination was apparent at each of the corresponding positions and thesequence could be determined. Given the constraints of Sangersequencing, the proofreading version of CORE3 could not be used,however, direct RNA sequencing should be adaptable to single moleculesequencing platforms (such as Pacbio's SMRT sequencing system), allowingproofreading of the RNA and eliminating the biases created in cDNAsynthesis and subsequent amplification.

By utilizing the RTCSR approach, an archaeal Family-B polymerase wasmorphed into a reverse transcriptase—establishing a family of reversetranscriptases entirely unbranched from natural RTs. The engineeredpolymerase can polymerize over long RNA templates with high accuracy andat elevated temperatures. High fidelity reverse transcription willenable more accurate understanding for many RNA processes. Mutations inRNA have been detected in many disease states, including cancer. Theprecise identification of rare somatic mutations in tissues are likelyto drive a better understanding of the disease process and diagnostictools. As RNA sequencing tools become more sophisticated, high fidelityreverse transcriptases will play a substantial role. The ability of theFamily-B reverse transcriptase to perform RNA sequencing without theneed to first create a cDNA library may become an invaluable tool forunderstanding the transcriptome at deeper levels.

The CORE3 polymerase reveals that high fidelity reverse transcription ispossible, and further supports that low fidelity reverse transcriptioncould be an adaptive aspect of retroviruses given that they have beenshown to have enormous potential to evolve in response to selectivepressures, such as the immune response (Wei et al., 1995). This islargely attributed to the vast diversity of the viral infection, formingwhat is often referred to as a quasi species (Lauring et al., 2010;Eigen, 1971). This may confirm the notion that low fidelity reversetranscription is adaptive or maybe even essential for retroviralpopulations. The introduction of high fidelity reverse transcriptasesinto retroviruses, perhaps limiting the genetic diversity available bylowering mutation rates, may serve as a mechanism to make attenuatedvaccines safer.

Example 2 Embodiment of Directed Evolution of Thermostable DNAPolymerases

The disclosure provides methods for producing derivative enzymes from aparent enzyme, wherein the derivative comprises one or more activitiesthat is lacking in the parent enzyme. The methods may utilize steps thatmodify the parent enzyme by random means and/or by targeted means ofmodification. Embodiments of the disclosure include modifications to aparent DNA polymerase that lacked reverse transcriptase activity.

In specific embodiments, methods for generating enzyme derivativesemploy a variation of the compartmentalized self-replication (CSR)method (Ghadessy et al. (2001); EP 1317539B). The CSR method is designedaround the directed evolution of thermostable DNA polymerases. In CSR,primers are designed which flank the polymerase gene. Upon thermocycling(PCR), the polymerase enzyme will copy their own genes. In the presentdisclosure, the method was adapted to allow evolution of alternativebases or sugars in the template strand. Specifically, in the presentvariation of CSR, the primer design is modified from known CSR methodsto enable the directed evolution of reverse transcriptases. The primersare designed such that a variable number of RNA bases are present in theprimer. After the first cycle of PCR, the primers become templates forsubsequent cycles. The method is designed such that only polymerasescapable of reverse transcription are recovered. Increasing the number ofRNA bases in the primer increase the stringency of reverse transcriptaseactivity. Primers can be composed entirely of RNA to allow maximumstringency (see FIG. 12).

Example 3 Exemplary Materials and Methods

The present example provides examples of materials and methods forembodiments of the disclosure.

Initial Reverse Transcription Test for Polymerases—

30 pmol of 5′ fluorescein labeled primer (25FAM) were annealed with 30pmol of template (TEMP.A.DNA/1RNA/5RNA) and 0.4 μg of polymerase by heatdenaturation at 90° C. for 1 minute and allowing to cool to roomtemperature. Reactions were initiated by the addition of “start” mixwhich contained (50 mM Tris-HCl (pH8.4), 10 mM (NH₄)₂SO₄, 10 mM KCl, 2mM MgSO₄ and 200 μM dNTPs. MMLV polymerase was treated according tomanufacturer's recommendations (New England Biolabs). Reactions wereincubated for 2 minutes at 68° C. until terminated by the addition ofEDTA to a final concentration of 25 mM. The labeled primer was removedfrom the template strand by heating sample at 75° C. for 5 minutes in 1×dye (47.5% formamide, 0.01% SDS) and 1 nmol of unlabeled BLOCKERoligonucleotide (to competitively bind the template strand). Sampleswere run on a 20% (7 M urea) acrylamide gel.

Reverse Transcription CSR (RTCSR)—

KOD polymerase libraries were created through error prone PCR unlessotherwise indicated to have a mutation rate of ˜1-2 amino acid mutationsper gene. Libraries were cloned into tetracycline inducible vector andelectroporated into DH10B E. coli. Library sizes were maintained with atransformation efficiency of at least 10⁶, but more typically 10⁷-10⁸.Overnight library cultures were seeded at a 1:20 ratio into fresh 2×YTmedia supplemented with 100 μg/mL ampicillin and grown for 1 hour at 37°C. Cells were subsequently induced by the addition ofanhydrotetracycline (typically at a final concentration of 200 ng/mL)and incubated at 37° C. for 4 hours. Induced cells (200 μL total) werespun in a tabletop centrifuge at 3,000×g for 8 minutes. The supernatantwas discarded and the cell pellet was resuspended in RTCSR mix: 1×Selection buffer (50 mM Tris-HCl (pH8.4), 10 mM (NH₄)₂SO₄, 10 mM KCl, 2mM MgSO₄), 260 μM dNTPs, 530 nM forward and reverse RNA containingprimers (detailed in Supplementary Table 1). The resuspended cells wereplaced into a 2 mL tube with a 1 mL rubber syringe plunger and 600 μL ofoil mix (73% Tegosoft DEC, 7% AbilWE09 (Evonik), and 20% mineral oil(Sigma-Aldrich)). The emulsion was created by placing the cell and oilmix on a TissueLyser LT (Qiagen) with a program of 42 Hz for 4 minutes.The emulsified cells were thermal-cycled with the program: 95° C.—3 min,20×(95° C.—30 sec, 62° C.—30 sec, 68° C.—2 min). Emulsions were brokenby spinning the reaction (10,000×g-5 min), removing the top oil phase,adding 150 μL of H₂O and 750 μL chloroform, vortexing vigorously, andfinally phase separating in a phase lock tube (5Prime). The aqueousphase was cleaned using a PCR purification column which results inpurified DNA, including PCR products as well as plasmid DNA.Subamplification with corresponding outnested recovery primers ensuresthat only polymerases that reverse transcribed are PCR amplified.Typically this is achieved by addition of 1/10 the total purifiedemulsion using Accuprime Pfx (ThermoFisher) in a 20 cycle PCR, howeverchallenging rounds of selection could require increasing the input DNAor cycle number to achieve desired amplification.

Cloning and Purification of Polymerase Variants—

Escherichia coli DH10B and BL21 (DE3) strains were used for cloning andexpression, respectively. Strains were maintained on either Superior or2XYT growth media. Polymerases were cloned into a modified pET21 vectorusing NdeI and BamHI sites. Overnight cultures of BL21 (DE3) harboringeach of the variants were grown overnight in Superior broth at 37° C.Cells were then diluted 1:250, and protein production was induced with 1mM IPTG during mid-log at 18° C. for 20 hrs. Harvested cells wereflash-frozen and lysed by sonication in 10 mM phosphate, 100 mM NaCl,0.1 mM EDTA, 1 mM DTT, 10% glycerol, pH 7 (Buffer A). Cleared celllysates were heated at 85° C. for 25 min, cooled on ice for 20 minutes,and filtered (0.2 μm). The filtrate was then passed over a DEAE column,immediately applied to an equilibrated heparin column, and eluted alonga sodium chloride gradient. Polymerase fractions were collected anddialyzed into Buffer A. Enzymes were further purified using an SP columnand again eluted along a salt gradient. Pooled fractions were thenapplied to a SEPHADEX® 16/60 size exclusion column (GE Healthcare),concentrated, and dialyzed into storage buffer (50 mM Tris-HCl, 50 mMKCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Non-idet P40, 0.1% Tween20, 50%glycerol, pH 8.0). Working stocks were made at 0.2 mg/mL.

PCR Proofreading Assay—

50 μL PCR reactions were set up with a final concentration of 1× AssayBuffer (60 mM Tris-HCl (pH8.4), 25 mM (NH₄)₂SO₄, 10 mM KCl), 200 μMdNTPs, 2 mM MgSO₄, 400 nM (PCRTest.F/PCRTest.R) or(PCRTest.DiDe.F/PCRTest.DiDe.R) forward and reverse primers, 20 ng ofpTET.KOD plasmid and 0.2 μg polymerase. Reactions were thermal-cycledusing the following program: 95° C.—1 min, 25×(95° C.—30 sec, 55° C.—30sec, 68° C.—2 min 30 sec).

Primer Extension Assay—

10 pmol of 5′ fluorescein labeled primer (RT.Probe or RT.Probe.3ddc)were annealed with 50 pmol of template RNA or DNA (RT.RNA.TEMP andRT.DNA.TEMP, respectively) and 0.4 μg of polymerase by heat denaturationat 80° C. for 1 minute and allowing to cool to room temperature.Reactions were initiated by the addition of “start” mix which contained(1× Assay Buffer, 2 mM MgSO₄ and 200 dNTPs. Reactions were incubated for10 minutes at 68° C. until terminated by the addition of EDTA to a finalconcentration of 25 mM. The labeled primer was removed from the templatestrand by heating sample at 75° C. for 5 minutes in 1× dye (47.5%formamide, 0.01% SDS) and 1 nmol of unlabeled RT.bigBlockeroligonucleotide (to competitively bind the template strand). Sampleswere run on a 20% (7 M urea) acrylamide gel.

Reverse Transcriptase Fidelity (SSCS)—

Templates for SSCS were prepared by first strand reverse transcriptionor primer extension (plasmid DNA template) with barcoded primer.Polymerization reactions were carried out according to manufacturer'srecommendations for recombinant MMLV (New England Biolabs). Forexperimental polymerases, reverse transcription or primer extension wasperformed in 1× Assay Buffer, 200 μM dNTPs, 1 mM MgSO₄, 400 nM barcodedreverse primer (HSP.seqBAR.R or pol2.SeqBar.R), 40 units RNasin Plus,0.2 μg polymerase, and template (1 μg Human heart total RNA or 1 ngplasmid). Reactions were incubated at 68° C. for 30 minutes (cDNAsynthesis) or 2 minutes for DNA primer extension. Single strandedproducts were PCR amplified using Accuprime Pfx polymerase(ThermoFisher) with nextSeq.R and corresponding indexed forward primer.Samples were submitted for ILLUMINA® MISEQ® PE 2×250 sequencer.

Targeted DNA sequencing reads were aligned and grouped based on uniquemolecular barcodes tagging individual reverse transcription events usingustacks (v1.35). Using a modified version of the single strand consensussequence program (SSCS) (Schmitt et al., 2012), only groups containingthree or more reads were analyzed. From these reads, a consensussequence was built if more than sixty-six percent of the bases at eachposition were in agreement, otherwise the base was called as N anddisregarded in the remaining analysis. Consensus reads were then alignedto the reference sequence using BWA-MEM (v0.7.7) (Li, 2013), and singlenucleotide variants and indels were identified. The polymerase fidelitywas calculated as the sum of indels and erroneous bases as a fraction ofthe total number of aligned bases.

RTPCR Assay—

50 μL reverse transcription PCR (RTPCR) reactions were set up on icewith the following reaction conditions: 1× Assay Buffer, 1 mM MgSO₄, 1 MBetaine (Sigma-Aldrich), 200 μM dNTPs, 400 nM reverse primer, 400 nMforward primer, 40 units RNasin Plus (Promega), 0.2 μg polymerase and 1μg of Total RNA from Jurkat, Human Spleen or E. coli (Ambion). Primersets used: PolR2A (PolII.R, PolII.F1/F2/F4), p532 (p532.R,p532.F1/F2/F5), rpoC (rpoC.R, rpoC.F1/F2/F4). Reactions werethermal-cycled according to the following parameters: 68° C.—30 min,25×(95° C.—30 sec, 68° C. (63° C. for rpoC)—30 sec, 68° C.—30 s/kb).

Single Nucleotide Incorporation Kinetics—

Duplexes (DNA:DNA or DNA:RNA) were assembled by combining equimolaramounts of a DNA 25-mer (5′-CCCTCGCAGCCGTCCAACCAACTCA-3′) (SEQ ID NO. 8)and DNA or RNA 36-mer (3′-GGGAGCGTCGGCAGGTTGGTTGAGTGCCTCTTGTTT-5′) (SEQID NO. 9) in 10 mM Tris-HCl, 0.1 mM EDTA (pH 8.0). Solutions were heatedto 95° C. for 5 min, slowly cooled to 60° C. for 10 min, and then cooledto room temperature for 15 minutes. Reactions (100 μL) consisting ofassay buffer, 1 mM MgSO4, and 500 nM duplex were initiated by variableamounts of α-P32-dCTP (0.003-400 μM), which was diluted 1:400 inunlabeled dCTP. Reactions were allowed to proceed 3-14 minutes. 10 μLaliquots were quenched by the addition of EDTA (0.25 M finalconcentration) in 15-120 s intervals. Aliquots (2 μL) were spotted onDE81 filter paper and washed 6 times in 5% NaH₂PO₄ (pH 7), 2 times inddH₂O and finally in 95% EtOH. Dried filter paper was exposed for 24 hrsand imaged on a STORM scanner. Initial rates were obtained by analysisusing Fiji (Image J). Kinetic parameters were determined by non-linearregression using SigmaPlot10.

RNA Sequencing and Analysis—

RNA from U87MG glioblastoma cells (ATCC® HTB-14) were harvested usingtrizol LS following manufacturer's instructions (10296-028, Thermofisher scientific). Ribosomal RNAs were then removed from the RNAsamples using RIBO-ZERO® rRNA removal kit (MRZH11124, Epicentre) andcleaned using RNEASY® MINELUTE® Cleanup Kit (Qiagen). rRNA depleted RNAswere fragmented using NEBNEXT® Magnesium RNA Fragmentation Module(E6150S, NEB) to 200-300 bp size range followed by kinase treatment toprepare for adaptor ligation. ILLUMINA® libraries were prepared usingNEBNEXT® Multiplex Small RNA Library Prep kit (E7580, NEB) and sizeselected to remove adaptor dimers using AMPURE® XP beads. Six ILLUMINA®libraries were prepared from the same pool of RNA using experimentalreverse transcriptases and PROTOSCRIPT® II Reverse Transcriptase fromthe library prep kit. RNASeq libraries were sequenced on ILLUMINA®HISEQ® 2000 sequencer, 2×100 bp by the genome sequencing and analysisfacility at the University of Texas at Austin.

The evaluation of RNA-seq quality control metrics was performed viaRNA-SeQC (v1.1.8) (DeLuca et al., 2012). For transcript abundanceanalysis, fpkm values were generated through the cufflinks/cuffnormpipeline (v2.2.1) (Trapnell et al., 2012) and transformed both by log 2and to fit the range [−3,3].

RNA Sanger Sequencing—

Sanger sequencing reactions were set up by preparing 1× Assay Buffer, 1mM MgSO₄, 10 pmol RT.Probe, 50 pmol SangerGATC Template, 0.4 ugCore3exo-, and 50 μM dNTPs. For the indicated terminator nucleotide, a25:1 ratio of 3′dideoxy terminator to unmodified NTP was used. Reactionswere thermal cycled 6×(68° C.—20 sec, 85° C.—5 sec). Reactions wereterminated by the addition of EDTA to a final concentration of 25 mM.The labeled primer was removed by heating sample at 75° C. for 5 minutesin 1× dye (47.5% formamide, 0.01% SDS) and 1 nmol of unlabeledSangerBlocker oligonucleotide.

Example 4 Sequencing of Pairs VH and VL Sequences from B Cells

Isolation of Total B Cells—

Frozen PBMCs (10 million cells in 1 mL) were thawed at 37° C.,resuspended in 50 mL of RPMI 1640 (Lonza) supplemented with 10% FetalBovine Serum, 1× non-essential amino acids, 1× sodium pyruvate, 1×glutamine, 1× penicillin/streptomycin, and 20 U/mL DNAse I, andrecovered via centrifugation (300 g for 10 min at 20° C.). The cellswere then resuspended in 4 mL of RPMI and allowed to recover at 37° C.for 30 min. The cells were diluted with 10 mL of cold MACS buffer (PBSsupplemented with 0.5% BSA and 2 mM EDTA), collected by centrifugation(300 g for 10 min at 4° C.), and depleted of non-B cells using the HumanMemory B Cell Isolation Kit with an LD column (Miltenyi Biotec) as perthe manufacturer's instructions. This yielded 400,000-500,000 B cellsper vial.

Amplification of the Paired VH:VL Repertoire—

The paired VH and VL sequences were determined using a custom designedaxisymmetric flow focusing device (DeKosky et al., 2016) that iscomprised of three concentric tubes. Total B cells were suspended in 6mL of cold PBS and passed through the innermost tube at a rate of 0.5mL/min. Oligo d(T)₂₅ magnetic beads (1 μm diameter at a concentration of45 μL beads/mL solution; NEB) were washed, subjected to focusedultrasonication (Covaris) to dissociate any aggregates, resuspended in 6mL of lysis buffer (100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA, 1%Lithium dodecyl sulfate (LiDS), 5 mM DTT), and passed through the middletube at a rate of 0.5 mL/min. The outer tubing contained an oil phase(mineral oil containing 4.5% Span-80, 0.4% Tween-80, and 0.05% TritonX-100; Sigma-Aldrich) flowing at 3 mL/min. The cells, beads, and lysisbuffer were emulsified as they passed through a custom designed 120 μmdiameter orifice, and were subsequently collected in 2 mLmicrocentrifuge tubes. Each tube was inverted several times, incubatedat 20° C. for 3 minutes, and then placed on ice. Following thecollection phase, emulsions were pooled into 50 mL conicals, andcentrifuged (4,000 g for 5 min at 4° C.). The mineral oil (upper phase)was decanted, and the emulsions (bottom phase) were broken withwater-saturated cold diethyl ether (Fischer). Magnetic beads wererecovered following a second centrifugation step (4,000 g for 5 min at4° C.) and resuspended in 1 mL of cold Buffer 1 (100 mM Tris pH 7.5, 500mM LiCl, 10 mM EDTA, 1% LiDS, 5 mM DTT). The beads were then seriallypelleted using a magnetic rack, and washed with the following buffers: 1mL lysis buffer, 1 mL Buffer 1, and 0.5 mL Buffer 2 (20 mM Tris pH 7.5,50 mM KCl, 3 mM MgCl). The beads were split into two aliquots, and eachwas then pelleted one final time and resuspended in an RT-PCR mixture(DeKosky et al., 2016) containing VH and VL Framework Region 1 (FR1)linkage primers or VH and VL leader peptide (LP) linkage primers andeither the CORE3 enzyme “RTX” or a conventional reverse transcriptase“Quanta”. The RT-PCR mixtures were then added dropwise to 9 mL ofchilled oil phase in an IKA dispersing tube (DT-20, VWR) and emulsifiedusing an emulsion dispersing apparatus (ULTRA-TURRAX® Tube Drive; IKA)for 5 min. The emulsions were aliquoted into 96-well PCR plates (100uL/well), and subjected to RT-PCR under the following conditions: 30 minat 55° C. followed by 2 min at 94° C.; 4 cycles of 94° C. for 30 s, 50°C. for 30 s, 72° C. for 2 min; 4 cycles of 94° C. for 30 s, 55° C. for30 s, 72° C. for 2 min; 32 cycles of 94° C. for 30 s, 60° C. for 30 s,72° C. for 2 min; 72° C. for 7 min; held at 4° C.

Following RT-PCR, the emulsions were collected in 2 mL microcentrifugetubes and centrifuged (16000 g for 10 min at 20° C.). The mineral oil(upper phase) was decanted, and water-saturated ether was used to breakthe emulsions. The aqueous phase (containing the DNA) was extractedthree times by sequentially adding ether, centrifuging the samples(16000 g for 30 s at 20° C.), and removing the upper ether phase. Traceamounts of ether were removed using a SpeedVac for 30 min at 20° C. TheDNA amplicons were purified using a silica spin column (Zymo Research)according to the manufacturer's instructions, and eluted in 40 μL H₂O.The two samples were then amplified through a nested PCR using PlatinumTaq (Life Technologies) under the following conditions: (FR1 primerderived sample) 2 min at 94° C., 32 cycles of 94° C. for 30 s, 62° C.for 30 s, 72° C. for 20 s; 72° C. for 7 min; held at 4° C.; (LP primerderived sample) 2 min at 94° C., 27 cycles of 94° C. for 30 s, 62° C.for 30 s, 72° C. for 20 s; 72° C. for 7 min; held at 4° C. Theamplicons, approximately 850 bp in length, were gel purified from 1%agarose using a gel extraction kit (Zymo Research) according to themanufacturer's instructions, and eluted in 20 μL H₂O.

To determine the full length VH and VL reads for antibody expressionstudies, the paired amplicon was subjected to an additional PCR usingNEBNEXT® high fidelity polymerase (NEB) to specifically amplify the fullVH chain and the full VL chain separately in addition to the pairedchains (Note: the paired reads sequence the entire J- and D-regions, andthe fragment of the V regions spanning FR2 to CDR3). Each sample wassplit into 5 reactions and subjected to the following PCR conditions: 30s at 98° C., X cycles of 98° C. for 10 s, 62° C. for 30 s, 72° C. for Ys; 72° C. for 7 min; held at 4° C. Finally, these sequences wereamplified one final time with TSBC compatible barcoding primersfollowing the protocol shown in, gel purified from 1% agarose using agel purification kit according to manufacturer's instructions, andsubmitted for paired-end ILLUMINA® next-generation sequencing. Theclustering of the resulting VH:VL pairs obtained by using the CORE3enzyme versus a conventional RT are shown in FIG. 13.

Example 5 Reverse Trascriptase and 2′ O-Methyl DNA Activity

Primer Extension Assay—

5 pmol of 5′ fluorescein labeled primer (RT.NoU.Probe) were annealedwith 12.5 pmol of template RNA (RT.NoU.Template) and 0.4 μg ofpolymerase by heat denaturation at 80° C. for 1 minute and allowing tocool to room temperature. For these studies the template RNA wasdesigned to lack “U” positions. Reactions were initiated by the additionof “start” mix which contained: 1× Assay Buffer, 1 mM MgSO₄ and 200 μMdNTPs. Reactions were incubated for 30 minutes at 68° C. The labeledprimer was removed from the template strand by heating sample at 75° C.for 5 minutes in 1× dye (47.5% formamide, 0.01% SDS) and 1 nmol ofunlabeled blocker oligonucleotide (to competitively bind the templatestrand). Samples were run on a 15% (7 M urea) acrylamide gel.

DNA Sequences

RT.NoU.Template (SEQ ID NO: 10)ACGCAAGGAGGCAAACGGAAAACAACGAGCAGGAGGGACGGCAGCGAGGG RT.NoU.Probe(SEQ ID NO: 11) CCCTCGCTGCCGTCCCTCCTG

Polymerase enzymes tested in the studies were based on the KOD enzyme,lacking exonuclease activity (by introduction of the D141A and E143Asubstitutions). This enzyme served as both the negative control andbackground for testing the effect of individual substitutions ofRNA-templated primer extension activity. The CORE3 enzyme (“RTX”) wasused as the positive control. The individual substitutions tested whereY384H, Y384I, V389I, Y493C, Y493L, I521L, E664K and G711V. Results ofthe studies in FIG. 14 show that each of the tested substitutions showedenhanced primer extension (RT) activity on a RNA template as compared tothe negative control, with the Y493L substitution showing the mostrobust activity.

Polymerase enzymes were also tested for ability to polymerize from a 2′O-methyl DNA template. Primer extension reactions were performed on aribose sugar analog [2′ O-methyl (Me) DNA] that indicated that CORE3(“RTX”) reverse transcription activity could extend alternativetemplates, although with lower efficiency, indicating a preference forRNA substrates (FIG. 15). However, the CORE3 enzyme “RTX” was still farmore efficient at using 2′-OMeDNA than the parental wild-type.

Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutionsand alterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

PATENTS AND PATENT APPLICATIONS

-   EP 1317539B

PUBLICATIONS

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1-84. (canceled)
 85. A recombinant Archaeal Family-B polymerasecomprising an amino acid sequence at least 80% identical to SEQ ID NO: 1and comprising an amino acid substitution corresponding to positionsR97, Y384, V389, Y493, F587, E664, G711, and W768 of SEQ ID NO:
 1. 86.The polymerase of claim 85, further comprising an amino acidsubstitution at a position corresponding to position 1521 in the aminoacid sequence shown in SEQ ID NO:1.
 87. The polymerase of claim 85,further comprising an amino acid substitution corresponding to positionN735 of SEQ ID NO:
 1. 88. The polymerase of claim 85, further comprisingan amino acid substitution corresponding to position 1521 and N735 ofSEQ ID NO: 1
 89. The polymerase of claim 85, further comprising an aminoacid substitution at a position corresponding to the group of F38, K118,M137, A490, R381, K466, T514, and E734, or a combination thereof, of SEQID NO:1.
 90. The polymerase of claim 85, further comprising an aminoacid substitution at a position corresponding to the group of F38, K118,M137, A490, R381, K466, T514, and E734, or a combination thereof, of SEQID NO:1.
 91. The polymerase of claim 85, wherein the amino acidsubstitution corresponding to position R97 is a methionine; to positionY384 is a phenylalanine, a leucine, an alanine, a cysteine, a serine, ahistidine, an isoleucine, a methionine, an asparagine, or a glutamineresidue; to position V389 is a methionine, a phenylalanine, a threonine,a tyrosine, a glutamine, an asparagine, or a histidine residue; to Y493is a leucine residue; to position F587 is a leucine residue; to positionE664 is a lysine, cystine, or phenylalanine residue; to position G711 isa leucine, a cysteine, a threonine, an arginine, a histidine, aglutamine, a lysine, or a methionine residue; and to position W768 is aarginine.
 92. The polymerase of claim 91, wherein the amino acidsubstitution corresponding to position Y384 is a histidine or anisoleucine residue; position V389 is an isoleucine or leucine residue;E664 is a lysine; and position G711 is a valine residue.
 93. Thepolymerase of claim 86, wherein the amino acid substitutioncorresponding to position 1521 is a leucine.
 94. The polymerase of claim87, wherein the amino acid substitution corresponding to position N735is lysine.
 95. The polymerase of claim 88, wherein the amino acidsubstitution corresponding to position 1521 is a leucine and positionN735 is lysine.
 96. The polymerase of claim 89, wherein the amino acidsubstitution at a position corresponding to F38 is a leucine orthreonine residue; at position K118 is an isoleucine residue; atposition M137 is an isoleucine or leucine residue; at position R381 is ahistidine residue; at position K466 is an arginine residue; at positionA490 is an threonine residue; at position T514 is an isoleucine; atposition F587 is an isoleucine or leucine; and at position E734 is anlysine.
 97. The polymerase of claim 85, wherein the polymerase hasproofreading activity.
 98. The polymerase of claim 85, wherein thepolymerase lacks proofreading activity.
 99. The polymerase of claim 85,wherein the polymerase transcribes a template that is RNA.
 100. Thepolymerase of claim 85, wherein the polymerase transcribes at least 10nucleotides from a RNA template.
 101. The polymerase of claim 85,wherein the polymerase has thermophilic activity.
 102. The polymerase ofclaim 85, wherein the polymerase transcribes a template that is2′-OMethyl DNA.
 103. A recombinant Archaeal Family-B polymerasecomprising an amino acid sequence at least 80% identical to SEQ ID NO: 1and comprising an amino acid substitution corresponding to positionsR97, Y384, V389, Y493, 1521, F587, E664, G711, N735, and W768 of SEQ IDNO:
 1. 104. A recombinant Archaeal Family-B polymerase comprising anamino acid sequence at least 80% identical to SEQ ID NO: 1 andcomprising an amino acid substitution corresponding to positions F38,R97, K118, M137, R381, Y384, V389, A490, K466, Y493, T514, 1521, F587,E664, G711, E734, N735, and W768 of SEQ ID NO:
 1. 105. The polymerase ofclaim 103, wherein the amino acid substitution corresponding to positionR97 is a methionine; to position Y384 is a phenylalanine, a leucine, analanine, a cysteine, a serine, a histidine, an isoleucine, a methionine,an asparagine, or a glutamine residue; to position V389 is a methionine,a phenylalanine, a threonine, a tyrosine, a glutamine, an asparagine, ora histidine residue; to Y493 is a leucine residue; to position 1521 is aleucine; to position F587 is a leucine residue; to position E664 is alysine, cystine, or phenylalanine residue; to position G711 is aleucine, a cysteine, a threonine, an arginine, a histidine, a glutamine,a lysine, or a methionine residue; to position N735 is lysine; and toposition W768 is a arginine.
 106. The polymerase of claim 103, whereinthe amino acid substitution corresponding to position F38 is a leucineor threonine residue; to position R97 is a methionine; to position K118is an isoleucine residue; to position M137 is an isoleucine or leucineresidue; to position R381 is a histidine residue; to position Y384 is aphenylalanine, a leucine, an alanine, a cysteine, a serine, a histidine,an isoleucine, a methionine, an asparagine, or a glutamine residue; toposition V389 is a methionine, a phenylalanine, a threonine, a tyrosine,a glutamine, an asparagine, or a histidine residue; to position K466 isan arginine residue; to position A490 is an threonine residue; toposition Y493 is a leucine residue; to position T514 is an isoleucine;to position 1521 is a leucine; to position F587 is an isoleucine orleucine residue; to position E664 is a lysine, cystine, or phenylalanineresidue; to position G711 is a leucine, a cysteine, a threonine, anarginine, a histidine, a glutamine, a lysine, or a methionine residue;to position E734 is an lysine; to position N735 is lysine; and toposition W768 is a arginine.